DON’T SAY IT WOULDN’T HAPPEN
a WORLD without EXTINCTION
The cloning revolution / By Steve Connor / 18 April 2008
After Dolly comes a new scientific technique that is being used to save a doomed species of the white rhino. Could this herald a world without extinction?
A revolutionary form of cloning is to be used as part of a last-ditch effort to save one of the world’s rarest animals – the northern white rhino – which is on the brink of extinction with only a few individuals left in the wild. British scientists are to spearhead an attempt to preserve the genes of a rhino in captivity by using a technique that mixes its skin cells with the embryos of a close cousin, the southern white rhino, which is not so endangered. The resulting offspring will be “chimeras” with a mixture of cells from both sub-species, but it is hoped that some of them will grow up to produce the sperm and eggs of the northern white rhino and so boost the animal’s dwindling gene pool.
If the pioneering experiment is successful, the biologists hope to extend the technique to a wide range of other endangered species whose populations in the wild are severely depleted as a result of hunting and habitat loss. Specialists at the Royal Zoological Society of Scotland and the University of Edinburgh are putting the plan together, with the help of conservationists in the field, who have warned the days of the northern white rhino are numbered with just three or four animals left in the grasslands of north-east Africa. Ian Wilmut, who led the team that cloned Dolly the sheep, is part of the research project and has stated that the new technique is more promising and practical than the cloning method he used in his famous breakthrough more than 10 years ago.
Professor Robert Millar, the director of the Medical Research Council’s Reproductive Sciences Unit at Edinburgh University, who is leading the study, said: “There are a lot of African animals under the threat of extinction. We want to protect their genomes, but you have to protect their habitats as well. This is one of the ways of dealing with the problem, especially when the animals get to such low numbers in the wild. It is a method we need to start to get into place as an insurance policy – it’s clearly do-able according to the laboratory work.”
Scientists plan to take small samples of skin from the few northern white rhinos kept in captivity, as well as any animals temporarily captured in the wild, and transform them into embryonic-like cells using a new genetic-engineering technique extensively tested on laboratory mice. The technique involves altering a few regulatory genes, which has the
effect of “reprogramming” the adult skin cells back to an embryonic state so that it can then develop into any of the specialised tissues of the body – including the germ-line cells that give rise to sperm and eggs. One scientist warned this week in an interview with The Independent that the technique of induced pluripotent stem (iPS) cells could even be used on human beings by maverick IVF doctors wanting to help infertile couples, because it has proved so easy to use on mice with few apparent side-effects.
Robert Lanza, the chief scientific officer of the American biotechnology company Advanced Cell Technology in Massachusetts, also said he is collaborating with Chinese scientists to use iPS stem cells on the giant panda as part of a conservation programme. “The technology could have enormous value in conservation biology. In fact, we have work currently underway using iPS cells to rescue endangered animals,” Dr Lanza said. “We also have an agreement with the Chinese Giant Panda Breeding Group to work with them to use reprogramming techniques to convert giant panda cells – skin or other tissue samples they have stored – into iPS cells in order to rescue genes that would otherwise be lost from the planet forever,” he added. The Medical Research Council’s human reproductive sciences unit is going to work closely with Edinburgh Zoo on breeding technologies that could be used to conserve endangered species, such as the African wild dog, the Ethiopian wolf and the pygmy hippo. A new body called the Institute for Breeding Rare and Endangered African Mammals has been set up in the Scottish capital to bring a number of scientists together to share experience and resources.
Paul de Sousa, a stem-cell specialist at Edinburgh University, said that all mammals appear to share the same genes that can be engineered to reprogramme skin cells to induce iPS cells and that it should be possible to use the technique on the northern white rhino. “No one has done this before, but I’m confident that it can be done. You’d aggregate the cells in an embryo and what you would create would be a chimera of the rhino,” Dr de Sousa said. Conservation biology experts said the problems associated with using the tissue of endangered animals are formidable. “If it’s going to work, it’s still a long way off… It’s going to be very difficult,” said Professor Bill Holt, of the Zoological Society of London.
Thomas Bernd Hildebrandt
emai : Hildebrand [at] izw-berlin [dot] de
or HOW to KNOCK UP an ELEPHANT
The Ticklish Trick of Inseminating an Elephant / 03 March 2005
A ticklish business, artificially inseminating an elephant. With the help of high-tech ultrasound and computer gear, special protective clothing, wheelbarrows and not a little cooperation from Chai, a 26-year-old Asian elephant, Woodland Park Zoo officials hope the complicated process led by two German scientists will result in the pachyderm giving again birth, as she did four years ago. Chai got pregnant by natural means last time around, but it wasn’t all candy and flowers. She had to endure the stress of getting shipped off to a zoo in Missouri, where some of her fellow elephants showed her hostility. She came home with scars and a few chunks missing from her ears.
There was even less romance this time, but at least she got to stay home at the zoo’s spacious elephant compound and house. Nearby was her calf, Hansa, who was born Nov. 3, 2000, becoming the first elephant ever delivered at the 100-year-old Seattle zoo. Setting the stage was no easy task. Dr. Thomas Hildebrandt, one of two world-renowned German scientists called in to help, wore a bicycle helmet, ultrasound imaging goggles and covered himself in plastic protective gear.
Beneath Hildebrandt, his colleague, Dr. Frank Goeritz, sat on a stool in front of a bank of computer screens, electronic equipment and a jumble of computer and power cords. With several zoo keepers helping, Hildebrandt inserted an ultrasound probe into the elephant’s rectum while Goeritz fed a light-emitting tube into a larger catheter that had been inserted into Chai’s ”vestibule.” The vestibule is just one feature of an elephant’s 10-foot-long reproductive tract that makes artificial insemination difficult. Inside it is a dime-sized vaginal opening, two false openings on either side, and the bladder’s much larger opening.
After hours of preparation, examination and a messy enema involving wheelbarrows of dung to make for a clearer ultrasound image, Hildebrandt and Goeritz succeeded in inseminating Chai Tuesday night. ”It went very well,” Hildebrandt told the Seattle Post-Intelligencer. ”We’ll see.” Dr. Nancy Hawkes, general curator at the Seattle zoo, said it will be another 15-16 weeks before an ultrasound can confirm if Chai is pregnant. If she is, there will be another 22 months of gestation, with a due date in December 2006 or January 2007.
Anatomy is just one of the hurdles to elephant reproduction. For starters, it isn’t easy to pin down exactly when they’re ovulating – a process Chai goes through only three times a year. There are no male elephants at the Seattle zoo. And because elephant semen can’t be frozen, fresh semen for Chai had to be collected and flown in from a
zoo elephant in Tulsa, Okla., and a donor in Los Angeles, a bull that works part-time in the film industry.
Some experts believe successful reproduction of the captive elephant population may be critical to the species’ long-term survival. The Asian elephant is as an endangered species, largely because of habitat destruction. ”Reproduction technology is increasingly important for saving species,” Hildebrandt said. He and his colleagues at the Berlin Institute for Zoo Biology and Wildlife Research apply their skills to many animals, such as the critically endangered Northern White Rhino.
Hildebrandt and Goeritz, nicknamed the ”Berlin Boys” in some circles, may hold the most promise for turning things around. They were responsible for 12 of the 17 successful elephant pregnancies achieved using artificial insemination in the past decade, and the others also used their approach, the P-I reported. Hildebrandt and his colleagues perfected the ultrasound technique of guiding the insemination process by performing autopsies on elephants that had been culled from herds in South Africa because of overpopulation in dwindling habitats. The German scientists also use the ultrasound for visualizing ovaries and other features of the elephant reproductive tract to make the timed rendezvous of elephant egg and sperm as close to perfect as possible.
Pulling species from the brink
by Charles Colville / 19 March 2007
There are only thirteen northern white rhinos left in the world. The species is hovering on the brink of extinction. But three men are pushing forward the frontiers of science to try to save them. Thomas Hildebrandt and his team, from the Berlin Institute of Zoo and Wildlife Research, are world leaders in using artificial reproduction to breed rare elephants, rhinos and even komodo dragons. Their work has never been more urgent. Throughout the history of Earth, 99% of all species which ever existed have disappeared. It is called the natural rate of extinction.
But now scientists think human activity is causing species to disappear at up to 10,000 times this rate. Many claim the last time this happened was 65 million years ago, when the dinosaurs died out. The great conservationist Richard Leakey has called it “the Sixth Mass Extinction”. Only one northern white rhino baby has been born in the last six years. Now, the Berlin team is working with six captive animals, at the Dvur Kralove Safari Park, 110km (70 miles) north-east of Prague, in the Czech Republic.
Last summer, they inseminated Fatu, one of only two fertile females in captivity. She had not been ovulating and needed hormone injections to get her cycle started. Months later, the results came in and unfortunately she did not get pregnant. “Despite the setback, we have to continue and we are very determined,” says Dr Hildebrandt. “We know that the work that we do is very important.”
Dr Hildebrandt is now convinced that artificial insemination alone will not save the species, so he is developing a ground-breaking IVF technique. Working with an international team from the Netherlands, Australia and China, he has already successful collected an oocyte, or egg, from a female of the more numerous southern white rhino species, at Western Plains Zoo, in New South Wales. The egg was fertilised in vitro, in a test tube, to produce an IVF rhino embryo. “Reproduction technology is increasingly important for saving species,” says Dr Hildebrandt, who knows that time is running out.
Later this year, the team will start to harvest eggs from the northern white rhino in the Czech Republic, and if all goes well, create baby northern whites. With so few northern white rhinos remaining, the researchers hope to use southern white rhinos as surrogate mothers. Dr Hildebrandt and his colleague Frank Goeritz were brought up in the former East Germany. They both suffered under the former communist regime and were initially not allowed to attend university, because of their middle-class background. Instead, they had to work as porters in an agricultural vet college. However, Dr Hildebrandt persuaded the head of the institute to allow him to study for a degree. That is when he started work on artificially inseminating cattle.
Within a few years, the zoologist was working with wild animals. Such was his passion for the subject that when the Berlin wall came down in 1989, he was too busy inseminating rare animals at the East Berlin Zoo to join the millions of his compatriots crossing to the West. Since then, the team has travelled ceaselessly across the world. Zoos and conservation projects from Australia to California have requested their services to boost breeding programmes.
However, the German scientists often confront unexpected obstacles on their travels. Last October Dr Hildebrandt collected semen from a male elephant at Pittsburgh Zoo, to use for inseminating a female elephant 3,000km away in Salt Lake City. The semen had to be placed in carry-on baggage, to avoid it being exposed to extreme temperatures or cosmic rays. At the time, liquids could not be transported on American planes, following the attempted terrorist attacks on transatlantic planes in August 2006.
Initially, airport security refused to give the go ahead and the project appeared doomed. Only after the intervention of the head of Pittsburgh Zoo did airport security officials relent, and allow Dr Hildebrandt and his elephant semen on board the plane. But even then, Dr Hildebrandt, and the elephant semen, had to be escorted by a bodyguard through the airport. Happily, the semen arrived within the eight-hour deadline, just in time to inseminate Christy, the female elephant at Salt Lake City Zoo.
So far, Dr Hildebrandt and fellow zoologists, Frank Goeritz and Robert Hermes, have successfully created 19 successful elephant calves. They are helping to create a captive breeding programme so that zoos will not be dependent on animals captured from the wild. But their biggest challenge is the northern white rhino where the stakes are far higher. It is the second largest land mammal and has lived on Earth for 50 million years, but is now dependent on Dr Hildebrandt’s team for its survival.
VISIT PLEISTOCENE PARK
“Pleistocene Park in Sakha region in northern Siberia is an attempt by Russian researcher Sergey Zimov to reproduce the ecosystem that flourished during the last ice age, with hopes to back his theory that hunting, and not climate change, destroyed the wildlife. Russian scientists are restoring the old ecosystem with plants and animals that thrived in the region 10,000 years ago. Japanese and Russian scientists hope to clone woolly mammoths, and to re-introduce them to the park. However, they have yet to find intact mammoth DNA to use for cloning.
So far, the scientific crew has successfully introduced reindeer, moose, musk oxen and yakut horses to the region, and the introduction of American bisons (instead of the extinct steppe bisons) is ongoing. Future introductions include saiga antelopes, yaks and siberian tigers. Pleistocene Park is a 160 km2 scientific nature reserve (zakaznik), owned and administered by a non-profit corporation, Pleistocene Park Association, consisting of the ecologists from the Northeast Science Station in Chersky and the Grassland Institute in Yakutsk. The reserve is surrounded by a 600 km2 buffer zone that will be added to the park by the regional government, once animals have successfully established.”
Animals to be introduced to the park: Wolverine, Lynx, Amur Leopard, Asiatic Black Bear, Brown Bear, Siberian Tiger, Asian Lion or African Lion, Kodiak Bear, Muskox, American Bison, Moose, Reindeer, Elk, Wisent, Bactrian Camel, Llama or Vicuña, Yak, Saiga Antelope
THEN STOP by the RESTAURANT
Mammoths to Return? DNA Advances Spur Resurrection Debate
by Mason Inman / June 25, 2007
Today the only place to see woolly mammoths and people side-by-side is on The Flintstones or in the movies. But researchers are on the verge of piecing together complete genomes of long-dead species such as Neandertals and mammoths. (See a brief overview of human genetics.) So now the big question is, Will we soon be able to bring such extinct species back to life? Researchers are divided over how they might try to do this and whether it’s even feasible. (Related: “Woolly Mammoth Resurrection, ‘Jurassic Park’ Planned [April 8, 2005].)
At the core of this issue is DNA, which encodes the thousands of genes that tell cells how to build themselves and keep running. Researchers already have deciphered the complete gene sequences—or genomes—for many living species, including humans, dogs, and mice. (Related: “Dog Genome Mapped, Shows Similarities to Humans” [December 7, 2005].)
The DNA of long-extinct species can also be preserved—in bones or bodies found in dry caves or inside ice, for example. “Retrieval of DNA from ancient specimens is relatively easy now,” said Alan Cooper, of the University of Adelaide in Australia. Even though such DNA has degraded into thousands of small pieces, researchers can still read these fragments and piece together much of the original genetic instructions.
Dead to Return?
So many researchers think that assembling the genome of Neandertals (often spelled “Neanderthals”) or mammoths is just around the corner. A team led by Stephan Schuster and Webb Miller at Pennsylvania State University and Tom Gilbert at the University of Copenhagen is working on the genome of woolly mammoths preserved in the Siberian permafrost. “I think it’s definitely feasible” to assemble these genomes, said Eske Willerslev of the University of Copenhagen in Denmark. But “it’s going to be extremely hard work.”
Svante Paabo, at the Max Planck Institute in Leipzig, Germany, and his colleagues are aiming to assemble a Neandertal genome from bones preserved in arid caves. (Related: “Neandertal DNA Partially Mapped, Studies Show” [November 15, 2006].) http://news.nationalgeographic.com/news/2006/11/061115-neanderthal-dna.html
In a paper appearing this week in Proceedings of the National Academy of Sciences, Paabo says that only certain types of errors appear in such ancient DNA, paving the way for scientists to more easily anticipate and correct gaps in their knowledge. But ideas of resurrecting these animals “is for the most part science fiction,” Paabo argued. Cooper, of the University of Adelaide, agrees. “As far as I can see, it is not going to be practical,” he said.
That’s because researchers are reading little fragments of preserved DNA and guessing at what the original genetic instructions were, Cooper said. “You’re not actually physically putting the DNA together, and I can’t see any way of doing that feasibly,” Cooper said. In large part, the problem is that living animals package their DNA with proteins that help it wind up into chromosomes. This packaging is crucial to making the DNA work properly, Cooper argues. Willerslev, of the University of Copenhagen, said the only way he could see of bringing back an extinct species like a mammoth would be to find an extremely well-preserved cell. That’s extremely unlikely to happen, he added, because all parts of a cell break down over time, even in mammoths that have been encased in ice since they died. But, he said, researchers working on cloning have contacted him, wanting to get a hold of mammoth tissue so they could try to clone a mammoth. “I was surprised,” Willerslev said. “I thought it was completely ridiculous.” These cloning researchers are “pros,” he added. “But I don’t think they will find anything they can use” in the frozen tissue. Japanese researchers, meanwhile, have been searching for years for a preserved mammoth with intact sperm, which they say could be used to create a new mammoth. But researchers who work on ancient DNA think this is also unlikely. “This is not the way to do it,” said Hendrick Poinar, of McMaster University in Canada.
Recipe for Resurrection
Miller, of Pennsylvania State University, however argues that we should never say never. “Do they also say that synthesizing a virus will never be possible?” he asked. This was accomplished for the first time in 2005, when researchers reassembled the deadly 1918 flu from preserved tissue samples. “What about a bacterium? A yeast? A fruit fly?” Miller added. “I’m curious where the line can be drawn.” McMaster University’s Poinar has his own ideas of how researchers might revive mammoths and other species—and he thinks it’s only a matter of time before it’s possible. “It’s theoretically possible, and I think it’s going to be done at some point,” Poinar said.
He says that once you have the genome of a mammoth, you could compare it with the genome of its closest relative, the Asian elephant. (Related: “Woolly Mammoth DNA Reveals Elephant Family Tree” [Dec. 20, 2005].)
Then you could genetically engineer the elephant DNA, point by point, so that it matches the mammoth DNA. Then, by inserting this modified DNA into an elephant’s egg cell, and implanting it in an elephant’s womb, you could create a modified elephant that’s nearly identical to the original mammoth, Poinar says. Or it could become possible to make entire chromosomes from scratch. “I wouldn’t be surprised if, in ten years, you’d be able to synthesize chromosome-length DNA,” Poinar said. “Five years ago everybody was saying you’d never be able to sequence the genomes of extinct animals … but here we are. We’re not that far away now.”
But Poinar isn’t sure we should bring these extinct animals back. “The more poignant question is whether this should be done,” he said. “This needs to be discussed way in advance. And the time is now, because it’s going move very, very quickly.” It’s not clear where we’d put a herd of mammoths, for example, and the natural predators that once
hunted them—other than people—are also extinct, he added. “I can’t think of a good reason to do it, other than the ‘wow’ value.”
to EAT THEM, SILLY
GRILLED MAMMOTH STEAK with BRAISED POTATO and NORTHERN GREENS
IN VITRO MEAT
Scientists Flesh Out Plans to Grow (and Sell) Test Tube Meat
by Alexis Madrigal / 04.11.08
In five to 10 years, supermarkets might have some new products in the meat counter: packs of vat-grown meat that are cheaper to produce than livestock and have less impact on the environment. According to a new economic analysis (1st .pdf) presented at this week’s In Vitro Meat Symposium in Ås, Norway, meat grown in giant tanks known as bioreactors would cost between $5,200-$5,500 a ton (3,300 to 3,500 euros), which the analysis claims is cost competitive with European beef prices. With a rising global middle class projected by the UN to double meat consumption (2nd .pdf) by 2050, and livestock already responsible for 18 percent of greenhouse gases, the symposium is drawing a variety of scientists, environmentalists and food industry experts. “We’re looking to see if there are other technologies which can produce food for all the people on the planet,” said Anthony Bennett of the United Nations Food and Agricultural Organization. “Not only today but over the next 10, 20, 30 years.”
Rapidly evolving technology and increasing concern about the environmental impact of meat production are signs that vat-grown meat is moving from scientific curiosity to consumer option. In vitro meat production is a specialized form of tissue engineering, a biomedical practice in which scientists try to grow animal tissues like bone, skin, kidneys and hearts. Proponents say it will ultimately be a more efficient way to make animal meat, which would reduce the carbon footprint of meat products. “To produce the meat we eat now, 75 to 95 percent of what we feed an animal is lost because of metabolism and inedible structures like skeleton or neurological tissue,” Jason Matheny, a researcher at Johns Hopkins and co-founder of New Harvest, a nonprofit that promotes research on in vitro meat, told Wired.com. “With cultured meat, there’s no body to support; you’re only building the meat that eventually gets eaten.”
Researchers can currently grow small amounts of meat in the lab, and have even been able to get heart cells to beat in Petri dishes. Growing muscle cells on an industrial scale is the next step, scientists say. “That’s the goal and it seems pretty clear from this conference that it’s achievable,” said Matheny on Thursday by telephone from the symposium.
Scientists are working on a variety of cell culture procedures. The cutting edge of in vitro meat engineering is the attempt to get cells to grow as if they were inside a living animal. Meat like steak is a complex combination of muscle, fat and other connective tissue. Reproducing the complexity of muscle is proving difficult. “An actual whole muscle organ is not technically impossible,” said Bob Dennis, a biomedical engineer at both North Carolina State University and the University of North Carolina, who attended the conference. “But of all the tissue engineering applications it is by far the most difficult
While scientists are struggling to recreate filet mignon, they anticipate less trouble growing hamburger. “The general consensus is that minced meat or ground meat products — sausage, chicken nuggets, hamburgers — those are within technical reach,” Matheny said. “We have the technology to make those things at scale with existing technology.” At scale, in this case, would be thousands of tons per year, Dennis said. But once the meat is made, consumer acceptance is far from assured. What cultured meat will taste like is up in the air. Some scientists think it could be used to create novel foods that won’t be quite meat, but won’t quite be anything else either. “I was once at a conference of food designers and they really liked the idea that they were not bound to a certain product that we know,” said Stig Omholt, a professor at the Norwegian University of Life Sciences and chairman of the In Vitro Meat Consortium. “We could make novel products.”
But most of the trends in food run counter to high-tech meat production. Heirloom tomatoes, organic produce and free-range-raised meat that pack the aisles of Whole Foods harken to previous, lower-tech eras. None of the experts were sure if there is a large market of early adopters who want to eat test tube meat for environmental, health or ethical reasons. For all the talk of high-tech meat production, attendees of the first In-Vitro Meat Symposium didn’t put their stomachs where their mouths were. Instead of sampling early versions of in vitro meat, they stuck to local fare. “We had some excellent Norwegian salmon, which was very tasty,” Bennett said.
email : bob [at] bme [dot] unc [dot] edu
‘WHAT is CULTURED MEAT?’
“Cultured meat is meat produced in vitro, in a cell culture, rather than from an animal. The production of cultured meat begins by taking a number of cells from a farm animal and proliferating them in a nutrient-rich medium. Cells are capable of multiplying so many times in culture that, in theory, a single cell could be used to produce enough meat to feed the global population for a year. After the cells are multiplied, they are attached to a sponge-like “scaffold” and soaked with nutrients. They may also be mechanically stretched to increase their size and protein content. The resulting cells can then be harvested, seasoned, cooked, and consumed as a boneless, processed meat, such as sausage, hamburger, or chicken nuggets.
Why would anyone want to make cultured meat?
Cultured meat has the potential to be healthier, safer, less polluting, and more humane than conventional meat. Fat content can be more easily controlled. The incidence of foodborne disease can be significantly reduced, thanks to strict quality control rules that are impossible to introduce in modern animal farms, slaughterhouses, or meat packing plants. Inedible animal structures (bones, respiratory system, digestive system, skin, and the nervous system) need not be grown. As a result, cultured meat production should be more efficient than conventional meat production in its use of energy, land, and water; and it should produce less waste.
How does cultured meat taste?
Cultured meat contains the same muscle cells that form most meats. However, there are a number of technical obstacles, especially regarding texture, that have to be overcome before cultured meat can be a compelling substitute for conventional meat.
Where can I buy cultured meat?
Cultured meat is not yet commercially available.
When will cultured meat be commercially available?
Within several years, it may be possible to produce cultured meat in a processed form, like sausage, hamburger, or chicken nuggets, with modifications of existing technologies. Producing unprocessed meats, like steaks or pork chops, would involve technologies that do not yet exist and that may take a decade or longer to develop.
What is the source of nutrients used in cultured meat production?
In biomedical research, most cell cultures have used media made from the blood of cow fetuses. But researchers have now developed media made from plants and mushrooms.
Isn’t this food unnatural?
Cultured meat is unnatural, in the same way that bread, cheese, yogurt, and wine are unnatural. All involve processing ingredients derived from natural sources. Arguably, the production of cultured meat is less unnatural than raising farm animals in intensive confinement systems, injecting them with synthetic hormones, and feeding them artificial diets made up of antibiotics and animal wastes. At the same time, the conventional production of meat has led to a number of unnatural problems, including high rates of ischemic heart disease and foodborne illness, as well as soil and water pollution from farm animal wastes.
Is cultured meat genetically-modified?
There is nothing in the production of cultured meat that necessarily involves genetic modification. The cells that can be used to produce cultured meat are muscle and stem cells from farm animals. It is possible, however, that genetically-modifying a muscle cell would allow it to proliferate a greater number of times in culture, and may thus make cultured meat production more economical.
Are any animals killed in the production of cultured meat?
Not necessarily. It is possible to take a muscle biopsy from a live farm animal and culture the isolated muscle cells. If stem cells are used, these would likely be from a farm animal embryo.
How much will cultured meat cost?
Theoretically, cultured meat could afford higher resource and labor efficiencies, which could translate into lower costs, if cultured meat were produced at scale with an affordable medium. However, it is unlikely that cultured meat will soon compete with conventional meat in ordinary markets. There are technologies now found in virtually every household that originally cost too much for mass acceptance. Only after reductions in cost by several orders of magnitude were they mass—produced.
Who are you?
New Harvest is a nonprofit research organization working to develop new meat substitutes, including cultured meat. Our boards are comprised of scientists in biology, agriculture, public health, and medicine.”
“Despite its popularity, meat — both in its production and in its consumption — has a number of adverse effects on human health, environmental quality, and animal welfare. These include: diseases associated with the over-consumption of animal fats; meat-borne pathogens and contaminants; antibiotic-resistant bacteria due to the routine use of antibiotics in livestock; inefficient use of resources in cycling grains and water through animals to produce protein; soil, air, and water pollution from farm animal wastes; and inhumane treatment of farm animals. As meat consumption continues to increase, worldwide, these problems are now a global concern.
As a result, there is an increasing market for meat substitutes that have the taste and texture of meat, but do not cause the problems associated with conventional meat. Meat substitutes can be made from plants such as soybeans, peas, or wheat; mycoproteins; or from animal tissues grown in culture. There are several plant- and mycoprotein-based meat substitutes already on the market.
One novel line of research is to produce meat in vitro, in a cell culture, rather than from an animal. The production of such “cultured meat” begins by taking a number of cells from a farm animal and proliferating them in a nutrient—rich medium. Cells are capable of multiplying so many times in culture that, in theory, a single cell could be used to produce enough meat to feed the global population for a year. After the cells are multiplied, they are attached to a sponge-like “scaffold” and soaked with nutrients. They may also be mechanically stretched to increase their size and protein content. The
resulting cells can then be harvested, seasoned, cooked, and consumed as a boneless, processed meat, such as sausage, hamburger, or chicken nuggets.”
HUMANELY CLONED VS.
Test Tube Meat Nears Dinner Table
by Lakshmi Sandhana / 06.21.06
What if the next burger you ate was created in a warm, nutrient-enriched soup swirling within a bioreactor?
Edible, lab-grown ground chuck that smells and tastes just like the real thing might take a place next to Quorn at supermarkets in just a few years, thanks to some determined meat researchers. Scientists routinely grow small quantities of muscle cells in petri dishes for experiments, but now for the first time a concentrated effort is under way to mass-produce meat in this manner. Henk Haagsman, a professor of meat sciences at Utrecht University, and his Dutch colleagues are working on growing artificial pork meat out of pig stem cells. They hope to grow a form of minced meat suitable for burgers, sausages and pizza toppings within the next few years.
Currently involved in identifying the type of stem cells that will multiply the most to create larger quantities of meat within a bioreactor, the team hopes to have concrete results by 2009. The 2 million euro ($2.5 million) Dutch-government-funded project began in April 2005. The work is one arm of a worldwide research effort focused on growing meat from cell cultures on an industrial scale. “All of the technology exists today to make ground meat products in vitro,” says Paul Kosnik, vice president of engineering at Tissue Genesis in Hawaii. Kosnik is growing scaffold-free, self-assembled muscle. “We
believe the goal of a processed meat product is attainable in the next five years if funding is available and the R&D is pursued aggressively.”
A single cell could theoretically produce enough meat to feed the world’s population for a year. But the challenge lies in figuring out how to grow it on a large scale. Jason Matheny, a University of Maryland doctoral student and a director of New Harvest, a nonprofit organization that funds research on in vitro meat, believes the easiest way to create edible tissue is to grow “meat sheets,” which are layers of animal muscle and fat cells stretched out over large flat sheets made of either edible or removable material. The meat can then be ground up or stacked or rolled to get a thicker cut. “You’d need a bunch of industrial-size bioreactors,” says Matheny. “One to produce the growth media, one to produce cells, and one that produces the meat sheets. The whole operation could be under one roof.”
The advantage, he says, is you avoid the inefficiencies and bottlenecks of conventional meat production. No more feed grain production and processing, breeders, hatcheries, grow-out, slaughter or processing facilities. “To produce the meat we eat now, 75 (percent) to 95 percent of what we feed an animal is lost because of metabolism and inedible structures like skeleton or neurological tissue,” says Matheny. “With cultured meat, there’s no body to support; you’re only building the meat that eventually gets eaten.”
The sheets would be less than 1 mm thick and take a few weeks to grow. But the real issue is the expense. If cultivated with nutrient solutions that are currently used for biomedical applications, the cost of producing one pound of in vitro meat runs anywhere from $1,000 to $10,000.
Matheny believes in vitro meat can compete with conventional meat by using nutrients from plant or fungal sources, which could bring the cost down to about $1 per pound. If successful, artificially grown meat could be tailored to be far healthier than any type of farm-grown meat. It’s possible to stuff if full of heart-friendly omega-3 fatty acids, adjust the protein or texture to suit individual taste preferences and screen it for food-borne diseases. But will it really catch on? The Food and Drug Administration has already barred food products involving cloned animals from the market until their safety has been tested. There’s also the yuck factor. “Cultured meat isn’t natural, but neither is yogurt,” says Matheny. “And neither, for that matter, is most of the meat we eat. Cramming 10,000 chickens in a metal shed and dosing them full of antibiotics isn’t natural. I view cultured meat like hydroponic vegetables. The end product is the same, but the process used to make it is different. Consumers accept hydroponic vegetables. Would they accept hydroponic meat?”
Taste is another unknown variable. Real meat is more than just cells; it has blood vessels, connective tissue, fat, etc. To get a similar arrangement of cells, lab-grown meat will have to be exercised and stretched the way a real live animal’s flesh would. Kosnik is working on a way to create muscle grown without scaffolds by culturing the right combination of cells in a 3-D environment with mechanical anchors so that the cells develop into long fibers similar to real muscle. The technology to grow a juicy steak, however, is still a decade or so away. No one has yet figured out how to grow blood vessels within tissue. “In the meantime, we can use existing technologies to satisfy the demand for ground meat, which is about half of the meat we eat (and a $127 billion global market),” says Matheny.
MITIGATING MUSCLE ATROPHY
“Revere is thinking about how to grow meat without the animal. It’s a cool idea that’s been floating around in science fiction for a while now, but, well, of course it has problems, and Revere notes a couple:
‘The two biggest, as far as I can see from a quick perusal of the burgeoning literature, are finding a suitable nutrient to grow the cells in; and then growing tissue that has the proper texture for being a meat substitute. Animal meat is not just muscle cells but a complicated structure also containing connective tissue, blood and blood vessels, nerves and fat. Just growing up masses of identical cells isn’t sufficient. You have to reproduce an architecture.’
I see those two problems as aspects of one much bigger problem. Muscle doesn’t grow in isolation: it’s always in a solid environmental context. It’s made up of cells that respond to activity in a way that enhances performance for the organism, and incidentally promotes flavor and texture and bulk for the delectation of the carnivore. So what do you need to make edible muscle mass, beyond a sheet of myocytes in a culture dish (which, I suspect, would have the texture of slime and would not sell well in test markets)?
An architecture is right. You need connective tissue to form a framework and you need a rigid but motile structure to do work and exercise the growing muscle. Then, because you want a piece of muscle larger than a drop, you need a delivery system for nutrients: a circulatory system, with a pump. This muscle in a vat is going to need a skeleton and a heart.
When I teach physiology, one of the organs I emphasize is the liver. It’s amazing how important a liver is to just about everything: growth, digestion, physical performance, reproduction, the whole shebang. Our cultured muscle will need a liver equivalent to support it. Even if we get rid of the digestive system entirely and feed this muscle mass on delivered supplies of pure glucose, amino acids, and various cofactors and enzymes, the liver is a primary regulatory agent for those substances.
Then we need an immune system. A huge lump of cells growing in a bath of sugar and amino acids is bacterial heaven — it’s going to need major antibacterial/antiviral support.
The more I think about it, the more I think people are going at it backwards. We shouldn’t be thinking about building muscle from the cells up, to create a purified system to produce meat for the market, we should be going the other way, starting with self-sustaining meat producers and genetically paring away the less commercially viable
bits, like the brain. Instead of test-tube meat, we should be working on more efficient organisms that generate muscle tissue with the properties we want.
Guess what? Farmers have already been doing this! Look at the domestic cow and chicken and turkey: they’re far more brainless than their wild relatives, and have been reduced to as much stupidity and helplessness as possible, without compromising their ability to survive semi-autonomously and harvest nutrients from naturally occurring food sources. I don’t see all that much difference in the consequences between building up a functional meat producer from cells in a dish, and stripping down a functional meat producer from a line of domesticated animals. Both starting points are aiming at the same final result; I suspect that the top down procedure is more likely to achieve success in my lifetime.”
U.S. PATENT # 6835390
FIELD OF THE INVENTION
The field of the present invention relates to producing and harvesting meat products for consumption. In particular, it relates to tissue engineered meat for consumption.
BACKGROUND OF THE INVENTION
Meat products such as beef, pork, lamb, poultry, or fish are desirable products for food consumption. Meat products are currently produced from whole animals, which is a highly inefficient production method because a significant portion of all agriculturally produced grain is used for animal rather than human consumption. In the United States, for example, livestock feed accounts for approximately 70% of all the wheat, corn, and other grain produced. In addition, to produce one pound of beef, thousand of pounds of water are required for the animal to drink and to grow the livestock feed. Meanwhile, throughout the world, by some account, over 800 million people are malnourished and 50,000 people die of starvation every day.
Current meat production methods are also harmful to the environment. Rain forests are depleted at a rate of approximately 500 square feet of rain forest for every pound of beef to be grown. Likewise, modem techniques for fishing marine life have become so efficient that the oceans and lakes are over-fished. Species that were once common are
now endangered or extinct.
Current scientific efforts to address these problems have focused on increasing the effectiveness of breeding or growing livestock. For example, growth hormones have been used to make livestock grow faster and thus, consume less grain and water. Growth hormones are typically injected into the livestock, but new methods of introducing the growth hormone have also been developed using genetic engineering technologies such as transgenics or cloning of the whole animal. Current meat production methods, nonetheless, require water, grain, and land to raise livestock.
Another problem with current meat production methods involves food contamination. Every year, on average, each American becomes sick and 9,000 people die from something they have injested. To control food contamination, the government’s present strategy is to inspect meat during processing. The USDA and the FDA, however, rarely regulate the farms where pathogens originate because they lack the regulatory powers over the farms. Nonetheless, except for E. coli 0156:H7, dangerous bacteria are legally considered “inherent” to raw meat. Two of the “inherent bacteria,” however,–campylobacter and salmonella– account for 80% of all illnesses and 75% of all deaths from meat and poultry consumption.
In the poultry industry, for example, as much as 25% of broiler chickens and 45% of ground chickens are reportedly allowed to test positive for salmonella. The Center for Disease Control estimates that campylobacter infects 70% to 90% of all chickens. Campylobacter infections cause cramps, bloody diarrhea, and fever. Every year in the United States, campylobacter infection results in about 800 deaths. Infections with campylobacter may also lead to Guillian-Barre syndrome, a disease that requires intensive care for several weeks. The incidence of serious illness and death from these bacteria may increase as more antibiotic-resistant strains develop. This has caused some scientists to question the continued use of antibiotics as a feed supplement for livestock. Thus, there exists a need to produce meat products for consumption that is more efficient, safer, and healthier than the current methods of production.
SUMMARY OF THE INVENTION
The present invention is directed to tissue engineered meat products and methods for producing such meat products. In one embodiment of the invention, the meat product comprises muscle cells that are grown ex vivo. These muscle cells may be grown and attached to a support structure and may be derived from any non-human cells. In a preferred embodiment of the invention, the meat product is substantially free from any harmful microbial or parasitic contamination. Another embodiment of the invention is directed to a meat product comprising muscle cells and other cells such as fat cells or cartilage cells, or both, that are grown ex vivo together with the muscle cells. In another embodiment of the invention, the meat product comprises muscle cells that have been exposed to an electric or oscillating current.
GENERAL DESCRIPTION OF THE PREFERRED EMBODIMENT
Generally, meat products are taken from the muscles of animals. Butchers carve out corresponding cuts of beef, poultry, lamb, fish, or pork to be sold as steak, chicken breast, lamb chops, fish fillet, pork chops, etc. Meat products also include meat-product derivatives such as ground meat that may be processed into meatball, hamburger patty, fishball, sausage, salami, bologna, ham, etc. Meat products may also include muscle tissues or meat that has been seasoned or dried such as jerky.
One embodiment of the present invention involves a method for producing meat products that may be used for consumption. The method may include culturing muscle stem cells in vitro and allowing these cells to differentiate into specific types of muscle cells such as skeletal muscle cells or smooth muscle cells ex vivo. Muscle cells may be derived from any non-human animals consumed by humans such as mammals (e.g. cattle, buffalo, pigs, sheep, deer, etc.), birds (e.g. chicken, ducks, ostrich, turkey, pheasant, etc.), fish (e.g. swordfish, salmon, tuna, sea bass, trout, catfish, etc.),
invertebrates (e.g. lobster, crab, shrimp, clams, oysters, mussels, sea urchin, etc.), reptiles (e.g. snake, alligator, turtle, etc.), and amphibians (e.g. frog legs). Preferably, muscle cells are derived from pluri-potent embryonic mesenchymal stem cells that give rise to muscle cells, fat cells, bone cells, and cartilage cells. The muscle cells may also be derived from toti-potent embryonic stem cells such as cells from the blastocyst stage, fertilized eggs, placenta, or umbilical cords of these animals.
Muscle cells may be grown in culture into muscle tissues that are attached to a support structure such as a two or three-dimensional scaffold or support structure. The muscle cells may be grown on the two dimensional support structure such as a petri-dish forming several layers of cells that may be peeled and processed for consumption. Other examples of two dimensional support structures may include porous membranes that allow for diffusion of nutrients from culture media on one side of the membrane to the other side where the cells are attached. In this type of culture conditions, additional layers of cells may be achieved by exposing the cells to culture media from both sides of the membrane, i.e., cells received nutrients through diffusion from one side of the membrane and also from the culture media covering the cells growing on the membrane.
Muscle cells may also be grown on, around, or inside a three-dimensional support structure. The support structure may be sculpted into different sizes, shapes, and forms, as desired, to provide the shape and form for the muscle cells to grow and resemble different types of muscle tissues such as steak, tenderloin, shank, chicken breast, drumstick, lamb chops, fish fillet, lobster tail, etc. The support structure may be made from natural or synthetic biomaterials that are preferably non-toxic so that they may not be harmful if ingested. Natural biomaterials may include, for example, collagen, fibronectin, laminin, or other extracellular matrices. Synthetic biomaterials may include, for example, hydroxyapatite, alginate, polyglycolic acid, polylactic acid, or their copolymers. The support structure may be formed as a solid or semisolid support.
To provide for optimal cell and tissue growth, the support structure, preferably, has high porosity to provide maximal surface area for cell attachment. A three-dimensional support structure may also be molded to include a branched vascular network providing for delivery of nutrients into and shuttling out of metabolites from the cells at the inner mass of the meat product. In this particular embodiment, the branch vascular network may be edible by using non-toxic natural or synthetic biomaterials as mentioned above. Furthermore, the support structure may also include adhesion peptides, cell adhesion molecules, or other growth factors covalently or non-covalently associated with the support structure. Examples of the peptides include sequences such as Arg-Gly-Asp or Arg-Glu-Asp-Val. Niklason, L., et. al., Advances in Tissue Engineering of Blood Vessels and Other Tissues, Transplant Immunology, 5(4):303-306 (1997). This reference is hereby incorporated by reference as if fully set forth herein.
On the other hand, culture conditions for these muscle cells may include static, stirred, or dynamic flow conditions. For scaled up production, the preferred method is to use a bioreactor, which produces greater volume of cells and allows greater control over the flow of nutrients, gases, metabolites, and regulatory molecules. Furthermore, bioreactors may provide physical and mechanical signal such as compression to stimulate cells to produce specific biomolecules. Vacanti, J., et. al., Tissue Engineering: The Design and Fabrication of Living Replacement Devices for Surgical Reconstruction and Transplantation, Lancet, 354 Suppl. 1, pSI32-34 (1999). This reference is hereby incorporated by reference as if fully set forth herein.
In another embodiment of the invention, meat products derived from muscle cells grown ex vivo may include fat cells derived also from any non-human animals. Fattier meat is generally tastier, but with greater fat content comes greater risk of adverse health consequences such as heart disease. Thus, the ratio of muscle cells to fat cells may be
regulated in vitro to produce the meat products with optimal flavor and health effects. Regulation may be achieved by controlling the ratio of muscle and fat cells that are initially seeded in culture and/or by varying, as desired, the concentrations and ratio of growth factors or differentiation factors that act upon the muscle cells or fat cells.
In another embodiment of the invention, cartilage derived from chondrocytes may first form an underlying support layer or structure together with the support structure. Afterwards, muscle cells or fat cells, or both, may be seeded onto the chondrocyte layer. The interaction of muscle cells and chondrocytes may further provide the necessary regulatory signals required for tissue formation. Examples of meat products that have muscle cells and cartilage cells include chicken breast or pork ribs.
In a preferred embodiment of the invention, aseptic techniques may be used to culture the muscle cells resulting in meat products that are substantially free from harmful microbes such as bacteria, fungi, viruses, prions, protozoa, or any combination of the above. Harmful microbes may include pathogenic type microorganisms such as
salmonella, campylobacter, E. coli 0156:H7, etc. In addition, muscle cells grown in culture may be substantially free from parasites such as tapeworms that infect muscles of whole animals and that are transferred to humans through consumption of insufficiently cooked meat. Aseptic techniques may also be employed in packaging the meat products as they come off the biological production line. Such quality assurance may be monitored by standard assays for microorganisms or chemicals that are already known in the art. “Substantially free” means that the concentration of microbes or parasites is below a clinically significant level of contamination, i.e., below a level wherein ingestion would lead to disease or adverse health conditions.
In another preferred embodiment of the invention, the meat product derived from muscle cells grown ex vivo may be exposed to an electric or oscillating current. Unlike muscle tissues derived from whole animals, muscle tissues grown ex vivo or in vitro may have never been exercised (e.g. never been used to move a leg). Thus, exposing the muscle cells, muscle tissue, or the meat products in vitro to an electric or oscillating current may mimic exercise and increase the similarity in texture between meat grown ex vivo and meat derived from whole animals. The electric or oscillating current may also increase the growth rate of muscle cells ex vivo. The electric or oscillating current may be applied to the muscle stem cells or to the muscle cells after they have differentiated from the stem cells.
In another embodiment of the invention, other nutrients such as vitamins that are normally lacking in meat products from whole animals may be added to increase the nutritional value of the meat. This may be achieved either through straight addition of the nutrients to the growth medium or through genetic engineering techniques. For example, the gene or genes for enzymes responsible for the biosynthesis of a particular vitamin, such as Vitamin D, A, or the different Vitamin B complexes, may be transfected in the cultured muscle cells to produce the particular vitamin.
In another embodiment of the invention, regulatory factors, growth factors, or other gene products may also be genetically introduced into the muscle cells. These factors, known as myogenic regulatory factors (“MRFs”), may stimulate and regulate the growth of muscles in vivo, but may not normally be produced by muscle cells in vivo or in vitro. Thus, expressing myogenic regulatory factors in cultured muscle cells may increase the production of muscle cells in vitro.
In another embodiment of the invention, the meat products derived from muscle cells in vitro may include different derivatives of meat products. These derivatives may be prepared, for example, by grounding or shredding the muscle tissues grown in vitro and mixed with appropriate seasoning to make meatballs, fishballs, hamburger patties, etc. The derivatives may also be prepared from layers of muscle cells cut and spiced into, for example, beef jerky, ham, bologna, salami, etc. Thus, the meat products of the present invention may be used to generate any kind of food product originating from the meat of an animal.
The following examples illustrate how one skilled in the art may make use of the current invention to produce meat products in vitro. Methods in cell biology, cell culture, and immunohistochemistry that are not explicitly described in this disclosure have already been amply reported in the scientific literature.
This example illustrates the isolation of pluri-potent mesenchymal stem cells for use in producing meat products in vitro. Mesenchymal stem cells give rise to muscle cells (myocytes), fat cells (adipocytes), bone cells (osteocytes), and cartilage cells (chrondocytes). Mesenchymal stem cells may be dissected and isolated from embryonic tissues of any non-human animal embryos. In cattle, for example, embryonic mesenchymal tissues that are rich in pluri-potent muscle stem cells are preferably isolated from embryos at day 30 to 40 or earlier. Once dissected, the embryonic tissues may be minced into small pieces about one millimeter by one millimeter in size in phosphate buffered saline (“PBS”) pH 7.45. Five to ten pieces of the minced tissue may be incubated in 300 .mu.l of 0.25% trypsin and 0.1% EDTA in PBS for thirty minutes at 37.degree. C. with gentle agitation. Afterwards, the tissues may be allowed to settle on the bottom of the tube by gravity or gentle centrifugation. The supernatant containing the trypsin/EDTA solution may then be aspirated and replaced with 300 .mu.l of 0.1% collagenase in PBS for ten to thirty minutes at 37.degree. C. Colleganese digestion may be repeated for several cycles as desired. Depending of the viscosity of the solution because of DNA released from damaged cells, 40 .mu.l of DNase I at 1 mg/ml in PBS may be added to the collagenase solution in between cycles.
The reaction may be stopped by adding medium such as DMEM or Ham’s F-12, or both in 1:1 ratio, (Life Technologies, Rockville, Md.) that is supplemented with 10 mM Hepes, 2 mM L-glutamine (Sigma-Aldrich), 10-20% heat-inactivated fetal calf or bovine serum (Hyclone Laboratories, Logan, Utah), penicillin at 100 units/ml and streptomycin at 100 .mu.g/ml (“complete medium”). Cells may be completely dissociated by gently pipetting the tissues up and down followed by washing the cells in complete medium once or twice using a centrifuge. The cells may then be plated onto an appropriate-sized petri dish which may be coated with natural biomaterials (e.g. collagen, fibronectin, laminin, or other extracellular matrices) or synthetic biomaterials (e.g. hydroxyapatite, alginate, polyglycolic acid, polylactic acid, or their copolymers), or both, and may be grown at 37.degree. C. and equilibrated with 5% CO.sub.2.
After mesenchymal stem cells have been isolated, they may be enriched for myoblasts or muscle stem cells in culture. Initially, the cells may be differentially plated on different petri dishes after dissociation and washing as described in Example I. Using a 60 mm petri dish, the cells may first be incubated in complete medium for two to four hours. During this time, epithelial cells will tend to attach quickly to the petri dish while the myoblasts remain in the supernatant. The supernatant may then be collected and the myoblasts may be plated on a different petri dish coated with natural or synthetic biomaterials such as those mentioned in Example I. Myoblasts may be enriched by supplementing the growth media with growth factors such as skeletal muscle growth factor, prostaglandin F.sub.2.alpha. (“PGF.sub.2.alpha. “), and insulin-like growth factor I (“IGF-1”).
Further, myoblasts may be differentiated into specific myoctes or muscle cells by culturing the myoblasts in complete medium or in minimal media (e.g. complete medium less the fetal calf serum) supplemented with muscle specific growth or differentiation factors such as PGF.sub.2.alpha. at concentrations ranging from 24 pg/ml to 28 pg/ml, and insulin from 10.sup.-6 M to 10.sup.-5 M. To more closely mimic in vivo muscle cells, which are normally innervated by neuronal cells, the culture medium may also be supplemented with appropriate neurotransmitters such as acetylcholine.
Alternatively, myoblasts may be enriched from toti-potent embryonic stem cells. Toti-potent cells may be derived from in vitro fertilized eggs of an animal using in vitro fertilization techniques, from stem cells present in umbilical cords or placenta, or from Embryonic Stem (ES) cells isolated from cells at the blastocyst stage. ES cells, for example, may be collected, gently dissociated by trypsin, and cultured in vitro with recombinant leukemia inhibitory factor (Chemicon, San Diego, Calif.) and feeder cells such as growth arrested embryonic fibroblasts cells. These toti-potent cells may be treated with growth factors such as PGF.sub.2.alpha. or IGF-1 to induce the cells to
differentiate into myoblasts.
Using standard immunohistochemistry or in-situ hybridization techniques, myoblasts or myocytes (differentiated muscle cells) may be identified. Briefly, myoblasts or myocytes grown in culture may be transferred into glass slides coated with appropriate extracellular matrix as described above. These cells may be grown to the desired number and differentiation using the conditions described above. After a sufficient growth and differentiation period, the cells may be fixed with 4% formaldehyde. If intracellular antibody markers or nucleotide probes are to be used, the cell membranes may be permeabilized with 1% NP-40 or Triton-X. Antibodies against markers specific for myoblasts or myocytes such as myosin, titin, alpha-actinin available from Sigma.RTM. may be used to identify the cells using standard fluorescent immunohistochemistry techniques. Alternatively, single stranded RNA or DNA probes for these markers may also be used for in-situ hybridization.
In addition, when the muscle cells have been attached to a three dimensional support structure as disclosed below, they may be cryo-frozen, sectioned and identified using antibody markers such as antibodies against myosin, titin, 12101, troponin T, alpha actinin available from Sigma.RTM..
Two or three dimensional scaffolds or supports may be sculpted from natural biomaterials (e.g. collagen, fibronectin, laminin, or other extracellular matrix) or synthetic biomaterials (e.g. hydroxyapatite, alginate, polyglycolic acid, polylactic acid, and their copolymers), or both. Preferably, the three dimensional scaffolds are sculpted with branch pathways for nutrients and culture media to reach the internal mass of the forming muscle tissues. Examples of materials and construction methods for these scaffolds are provided by U.S. Pat. No. 5,686,091, entitled “Biodegradable Foams For Cell Transplantation”; U.S. Pat. No. 5,863,984, entitled “Biostable Porous Material Comprising Composite Biopolymers”; U.S. Pat. No. 5,770,417, entitled “Three-Dimensional Fibrous Scaffold Containing Attached Cells for Producing Vascularized Tissue in vivo;” and U.S. Pat. No. 5,916,265, entitled “Method of Producing a Biological Extracellular Matrix for Use as a Cell Seeding Scaffold and Implant.” These patents are hereby incorporated by reference as if fully set forth herein.
The support structure is preferably sculpted to different sizes, shapes, and forms to allow for growth of muscle tissues resembling different types of meat products such as steak, tenderloin, shank, chicken breast, drumstick, lamb chops, fish fillet, lobster tail, etc.
Adipocytes, chondrocytes, and ostooblasts are all capable of differentiating from pluri-potent mesenchymal stem cells or toti-potent embryonic stem cells. The stem cells may be isolated as described in Example I or III. The stem cells may be cultured in DMEM, or Ham’s F-12, or both in a 1:1 ratio. The medium may be supplemented with thyroid hormone, transferrin, insulin, as well as other growth factors, such as insulin-like growth factor (IGF), basic fibroblast growth factor, and growth hormone.
For adipocytes, differentiation may be achieved by treating the stem cells with bone morphogenetic proteins (“BMP”) such as BMP-4 and BMP-2, which are known to induce commitment to the adipocyte lineage. Ahrens et. al., Expression of human bone morphogenetic proteins-2 or -4 in murine mesenchymal progenitor C3H10T1/2 cells induces differentiation into distinct mesenchymal cell lineages, DNA Cell Biol., 12:871-880 (1993); Wang et. al., Bone Morphogenetic protein-2 causes commitment and differentiation in C3H10T1/2 and 3T3 cells. Growth Factors 9:57 (1993). These references are hereby incorporated by reference as if fully set forth herein.
In addition to BMPs, the differentiation of adipocytes may be enhanced with agonist of peroxisome proliferator-activated receptor gamma (“PPAR gamma”) such as BRL 49653 (rosiglitazone). Sottile and Seuwen, Bone morphogenetic protein-2 stimulates adipogenic differentiation of mesenchymal precursor cells in synergy with BRL 49653 9 (rosiglitzaone), FEBS Lett, 475(3):201-204 (2000). This reference is hereby incorporated by reference as if fully set forth herein.
In certain situations, myoblasts may even be induced to trans-differentiate into adipoblasts (adipocyte precursors) by treating myoblasts cells or muscle satellite cells with long-chain fatty acids (“LCFA”) or thiazolidinediones, or both Grimaldi et. al., Trans-differentiation of myoblasts to adipoblasts: triggering effects of fatty acids and thiazolidinediones, Prostaglandins Leukot Essent Fatty Acids, 57(1):71-75 (1997); Teboul et. al., Thiazolidinediones and fatty acids convert myogenic cells into adipose-like cells, J. Biol. Chem. 270(47):28183-28187 (1995). These references are hereby incorporated by reference as if fully set forth herein.
Thus, meat products with the desired amount of fat content may be produced by seeding and co-culturing muscle cells and adipocyte cells at a certain ratio. Alternatively, stem cells may be allowed to differentiate initially into myoblasts and then at a later time, LCFA or thiadolidinediones may be added at different concentrations and different exposure times to trans-differentiate the myoblasts into adipocytes as desired. Furthermore, the growth of muscle cells and fat cells may be regulated by controlling the concentration of the growth and differentiation factors. For example, if less fat cells are
desired in the final meat product, lesser concentrations of BMP factors may be added to the culture while a higher concentration of PGF.sub.2.alpha. and/or insulin may be added to promote muscle cell growth.
Chondrocytes or cartilage cells may also be isolated from an animal’s knee or rib cages. Using similar techniques as described in Example I, dissected tissue from the knee or rib cages may be minced, digested with collagenase, and washed with complete medium. The cells may then be differentially plated to increase the purity of chondrocyte cells.
It is known that chondrocytes differentiate in response to mechanical stress. Thus, preferably, the cells may be subjected to shear flow stress as described in U.S. Pat. No. 5,928,945, entitled “Application of Shear Flow Stress to Chondrocytes or Chondrocyte Stem Cells to Produce Cartilage,” which is hereby incorporated by reference as if fully set forth herein.
Chondrocytes may initially form a first layer of support cells in a three-dimensional scaffold. Myoblasts or adipocyte cells, or both, may then be seeded onto the chondrocyte layer and grown to the desired size. As such, the chondrocyte layer may provide additional adhesion or growth factors to the muscle cells.
Muscle cells grown in vitro differ from muscle cells grown in vivo in that in vivo cells are used during exercise or body movements. As muscles are used in vivo, muscle cells, in limbs for example, contract and relax in accordance with the movement of the limbs. Hence, to more closely mimic the growth of muscle cells in vivo, the cells grown in vitro may be exposed to an electric or oscillating current, or pulses of electric or oscillating current to contract the muscle cells. Electric probes may be immersed into the culture media to deliver mild current. Alternatively, the support structure may be coated with electrically conducting materials. Examples of electrically conducting materials and a method for coating them onto the support structure are described in U.S. Pat. No. 5,843,741, entitled “Method for Altering the Differentiation of Anchorage Dependent Cells on an Electrically Conducting Polymer,” which is hereby incorporated by reference as if fully set forth herein.
The preceding examples illustrate the procedures for producing meat products ex vivo. They are intended only as examples and are not intended to limit the invention to these examples. It is understood that modifying and combining the examples above do not depart from the spirit of the invention.