Similar processes occur in the process of distraction osteogenesis – a process I will interview my friend Cynthia Chang about in the next technique post, but also in normal bone fracture healing. In the case of my transplants I have seen firsthand the ability to transplant sponges completely devoid of any cells into a mouse, and harvest tissue weeks later that are not only full of cells, but have also created living tissue in these spaces. Where do these cells come from? And how to we get the right cells to come to our transplant?

In order to answer this question I’ve been studying the movement of human umbilical vein endothelial cells (HUVECs) because I want to encourage faster blood vessel formation in my scaffolds. The way that we do this is really simple. We grow cells on a petri dish in a special microscope that allows us to constantly monitor the cells while at the same time keeping them at the proper temperature and carbon dioxide level. Then, instead of taking a video, which would be enormous in file size, we take pictures every 5 minutes to see how far the cells have moved. This method, called “time-lapse” means that when we stitch all these photos together we essentially get a flip-book video of the cells movement – similar to the cartoon flip-books of childhood (I had a Wiley Coyote flipbook).

Once we have this flipbook of images, we can analyze it using the NIH free software ImageJ to individually track cells (Cell Tracking plugin). Then we can use another plugin (Chemotaxis Tool) to measure the directionality (i.e. whether movement is in one direction or truly random) as well as velocity. It’s very simple and gives a lot of data in a really short amount of time, so I would definitely recommend it to any PhD student out there who needs more in vitro data.

Basic Method:
1. Cells are plated at 0.075×106 per 35mm plate in normal medium and left overnight
2. The next day medium is changed to the desired conditions (I did growth factors in just basal medium vs. growth factors in addition to the full, serum-containing serum)
3. Dishes are placed in the time-lapse microscopes, and monitored over 48 hours, pictures every 5 minutes
4. Images are analyzed using ImageJ and statistics performed

Regenerative medicine is an upcoming technology field that may be capable of repairing and replacing damaged cells, tissues, and organs by using those that are specially grown. This technology has evolved by the combined efforts of scientists belonging to a diverse range of expertise, including, biology, genetics, physiology, nanotechnology, and pharmacology. Generally, regenerative medicine includes three sub-fields: tissue engineering, cell therapies, and healing therapies. While working in collaboration, tissue engineers focus on growing connective tissues and replacement organs for the body and their work is tested on animals before implementing it into humans, whereas cell therapy scientists try to stimulate reparation from the diseased tissue or organ itself by identifying and optimizing the factors that lead to self-reparation of the organ. Thereafter, healing therapists are involved in studying strategies to restore the functions of an implanted tissue or organ.

The general procedure includes cutting out a piece of the organ that is to be replaced, separating out the cells and placing them in fluid that allows them to multiply. After that, an animal organ is treated with mild detergent to remove all cells, leaving the basic support structure, called a scaffold. Consequently, patient’s cells are poured onto the scaffold, and the new organ is then placed in an incubator that simulates a human body, where the cells can grow and knit together. Finally, the organ is planted inside the patient’s body, and the scaffold slowly immaterializes, leaving only the regenerated organ.

A recent trial of this technology wasreported in The New York Times, which discussed about Sgt. Ron Strang, whose part of a left thigh was blown away by a roadside bomb in Afghanistan. The explosion and subsequent rounds of surgery left Sergeant Strang, 28, a Marine, with a huge divot in his upper thigh where the quadriceps muscle had been. He could move the leg backward, but with so much of the muscle gone he could not kick it forward. He could walk, but only awkwardly. Applying the technology of regenerative medicine, Sergeant Strang has now grown new muscle thanks to a thin sheet of material from a pig. More details about this may be readhere.

In brief, the thin sheet of material is an adipose tissue-derived cell growth scaffold, which, in layman term, is the extracellular matrix. Reconstruction of tissue, in general, includes provision of a matrix which serves as a guide for cells which grow along and between the fibres of the matrix. At present, biologic scaffolds composed of extracellular matrix (ECM) are utilized in numerous regenerative medicine applications to facilitate the constructive remodeling of tissues and organs. The mechanisms by which the host remodeling response occurs are not fully understood, but recent studies suggest that both constituent growth factors and biologically active degradation products derived from ECM play important roles.

This procedure was performed byDr. Peter Rubin, a plastic surgeon at the University of Pittsburgh Medical Center who is a leader of the said study. Dr. Rubin has also filed a patent application titled “DECELLULARIZED ADIPOSE CELL GROWTH SCAFFOLD” that has been allocated a publication numberWO/2011/087743. The details of the patent application can be accessedhere.

As it may be seen, the patent application primarily claims a method of preparing an adipose tissue-derived cell growth scaffold comprising washing a decellularized adipose tissue-derived cell growth scaffold with n-propanol, isopropanol or a mixture thereof. This method is further limited by use of various dependent claims, which includes preparing the decellularized adipose tissue- derived cell growth scaffold by: (a) digesting an adipose tissue sample with a proteinase; (b) washing the sample with a surfactant; (c) washing the sample with an emulsifier; (d) disinfecting the sample; (e) depyrogenating the sample; (f) drying the sample; (g) washing the sample in n-propanol, isopropanol or a mixture thereof; and (h) washing the sample in an aqueous solvent.

With reference to the report published in The New York Times, the claims directed to the referenced procedure would be claims 13-20. In accordance with claim 13, a method of growing cells comprises contacting cells with a cell growth scaffold as claimed in one of claims 1-10 and culturing the cells. This claim is further limited by various specific features of the procedure, such as, culturing the cells in vivo, applying the cell growth scaffold to a wound in a patient, applying the cell growth scaffold to a topical wound, and spraying the cell growth scaffold onto a topical wound.

The patent claims are further characterized to include the scaffold that is contained within a biocompatible cover to produce an implant, and the implant is implanted into a patient. The implant comprises cells, which may be chondrocytes, chondroprogenitor cells, adipose stem cells, mesenchymal stem cells, myocytes, precursor cells, progenitor cells, stem cells, differentiated cells.

With a view to analyze the patent trends in the field of regenerative medicine, it is important to note that the above-mentioned patent application pertains to the major international patent classification (IPC) of C12N 11/16, which generally relates to enzymes or microbial cells being immobilised on or in a biological cell. A quick search at the WIPO patent database reveals that there are 164 WIPO Patent Applications that include C12N 11/16 as primary patent classification. The following graph illustrates major players filing patents in this field.

Generally, the IPC (International Patent Classification) search is one of the most important patent fundamental search tools available to patent attorneys /patent agents and general public at large while conducting a patent search. Moreover, the International Patent Classification retrieves relevant patent specifications in order to establish the novelty of the technology in question and to evaluate the inventive step or non-obviousness of the inventive part of the patent application. The International Patent Classification system classifies patent applications and helps patent attorneys / patent agents to search patent specifications (patent applications, specifications of granted patents, utility models, and the like).

Furthermore, the International Patent Classification serves as an instrument for arrangement of the patent applications in an orderly format and provides an easy retrieval from the patent databases, and further provides a basis for selective dissemination of information to any person searching for patent resources.

The International Patent Classification acts as a very important tool for conducting state of the art patent search for given technical art in question and provides assessment of technological development in numerous technical fields. The state-of-the-art patent search is the broadest search and aims to locate patent publications in the specific technical field of invention and provide a valuable insight into what has been patented in a specific field of invention. Particularly, the state of the art patent search report assist in developing an IP strategy for long-term market advantage.

The International Patent Classification divides the world of technology into eight sections, which as illustrated below:

SECTION A — HUMAN NECESSITIES

SECTION B — PERFORMING OPERATIONS; TRANSPORTING

SECTION C — CHEMISTRY; METALLURGY

SECTION D — TEXTILES; PAPER

SECTION E — FIXED CONSTRUCTIONS

SECTION F — MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING

SECTION G — PHYSICS

SECTION H — ELECTRICITY

The above mentioned eight sections of the International Patent Classification has over 70,000 subdivisions. Each subdivision has a symbol consisting of Arabic numerals and letters of the Latin alphabet. For e.g., as discussed above, the IPC C12N11/16 relates to “Enzymes or microbial cells being immobilised on or in a biological cell”.

IPC classification symbols are made up of a letter denoting the IPC section (e.g. C), followed by a number (two digits) denoting the IPC class (e.g. C12), then a letter denoting the IPC subclass (e.g. C12N). A number (variable, 1-3 digits) denotes the IPC main group (e.g. C12N11). This is followed by a forward slash “/” and a number (variable, 1-3 digits) denoting the IPC subgroup (e.g. C12N11/16). To elaborate more on IPC classification symbols the following example can be considered:

C: CHEMISTRY; METALLURGY

C12: BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N: MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF

C12N11: Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof

C12N11/16: Enzymes or microbial cells being immobilised on or in a biological cell

C12N11/18: Multi-enzyme systems

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Regenerative medicine is an upcoming technology field that may be capable of repairing and replacing damaged cells, tissues, and organs by using those that are specially grown. This technology has evolved by the combined efforts of scientists belonging to a diverse range of expertise, including, biology, genetics, physiology, nanotechnology, and pharmacology. Generally, regenerative medicine includes three sub-fields: tissue engineering, cell therapies, and healing therapies. While working in collaboration, tissue engineers focus on growing connective tissues and replacement organs for the body and their work is tested on animals before implementing it into humans, whereas cell therapy scientists try to stimulate reparation from the diseased tissue or organ itself by identifying and optimizing the factors that lead to self-reparation of the organ. Thereafter, healing therapists are involved in studying strategies to restore the functions of an implanted tissue or organ.

The general procedure includes cutting out a piece of the organ that is to be replaced, separating out the cells and placing them in fluid that allows them to multiply. After that, an animal organ is treated with mild detergent to remove all cells, leaving the basic support structure, called a scaffold. Consequently, patient’s cells are poured onto the scaffold, and the new organ is then placed in an incubator that simulates a human body, where the cells can grow and knit together. Finally, the organ is planted inside the patient’s body, and the scaffold slowly immaterializes, leaving only the regenerated organ.

A recent trial of this technology wasreported in The New York Times, which discussed about Sgt. Ron Strang, whose part of a left thigh was blown away by a roadside bomb in Afghanistan. The explosion and subsequent rounds of surgery left Sergeant Strang, 28, a Marine, with a huge divot in his upper thigh where the quadriceps muscle had been. He could move the leg backward, but with so much of the muscle gone he could not kick it forward. He could walk, but only awkwardly. Applying the technology of regenerative medicine, Sergeant Strang has now grown new muscle thanks to a thin sheet of material from a pig. More details about this may be readhere.

In brief, the thin sheet of material is an adipose tissue-derived cell growth scaffold, which, in layman term, is the extracellular matrix. Reconstruction of tissue, in general, includes provision of a matrix which serves as a guide for cells which grow along and between the fibres of the matrix. At present, biologic scaffolds composed of extracellular matrix (ECM) are utilized in numerous regenerative medicine applications to facilitate the constructive remodeling of tissues and organs. The mechanisms by which the host remodeling response occurs are not fully understood, but recent studies suggest that both constituent growth factors and biologically active degradation products derived from ECM play important roles.

This procedure was performed byDr. Peter Rubin, a plastic surgeon at the University of Pittsburgh Medical Center who is a leader of the said study. Dr. Rubin has also filed a patent application titled “DECELLULARIZED ADIPOSE CELL GROWTH SCAFFOLD” that has been allocated a publication numberWO/2011/087743. The details of the patent application can be accessedhere.

As it may be seen, the patent application primarily claims a method of preparing an adipose tissue-derived cell growth scaffold comprising washing a decellularized adipose tissue-derived cell growth scaffold with n-propanol, isopropanol or a mixture thereof. This method is further limited by use of various dependent claims, which includes preparing the decellularized adipose tissue- derived cell growth scaffold by: (a) digesting an adipose tissue sample with a proteinase; (b) washing the sample with a surfactant; (c) washing the sample with an emulsifier; (d) disinfecting the sample; (e) depyrogenating the sample; (f) drying the sample; (g) washing the sample in n-propanol, isopropanol or a mixture thereof; and (h) washing the sample in an aqueous solvent.

With reference to the report published in The New York Times, the claims directed to the referenced procedure would be claims 13-20. In accordance with claim 13, a method of growing cells comprises contacting cells with a cell growth scaffold as claimed in one of claims 1-10 and culturing the cells. This claim is further limited by various specific features of the procedure, such as, culturing the cells in vivo, applying the cell growth scaffold to a wound in a patient, applying the cell growth scaffold to a topical wound, and spraying the cell growth scaffold onto a topical wound.

The patent claims are further characterized to include the scaffold that is contained within a biocompatible cover to produce an implant, and the implant is implanted into a patient. The implant comprises cells, which may be chondrocytes, chondroprogenitor cells, adipose stem cells, mesenchymal stem cells, myocytes, precursor cells, progenitor cells, stem cells, differentiated cells.

With a view to analyze the patent trends in the field of regenerative medicine, it is important to note that the above-mentioned patent application pertains to the major international patent classification (IPC) of C12N 11/16, which generally relates to enzymes or microbial cells being immobilised on or in a biological cell. A quick search at the WIPO patent database reveals that there are 164 WIPO Patent Applications that include C12N 11/16 as primary patent classification. The following graph illustrates major players filing patents in this field.

Generally, the IPC (International Patent Classification) search is one of the most important patent fundamental search tools available to patent attorneys /patent agents and general public at large while conducting a patent search. Moreover, the International Patent Classification retrieves relevant patent specifications in order to establish the novelty of the technology in question and to evaluate the inventive step or non-obviousness of the inventive part of the patent application. The International Patent Classification system classifies patent applications and helps patent attorneys / patent agents to search patent specifications (patent applications, specifications of granted patents, utility models, and the like).

Furthermore, the International Patent Classification serves as an instrument for arrangement of the patent applications in an orderly format and provides an easy retrieval from the patent databases, and further provides a basis for selective dissemination of information to any person searching for patent resources.

The International Patent Classification acts as a very important tool for conducting state of the art patent search for given technical art in question and provides assessment of technological development in numerous technical fields. The state-of-the-art patent search is the broadest search and aims to locate patent publications in the specific technical field of invention and provide a valuable insight into what has been patented in a specific field of invention. Particularly, the state of the art patent search report assist in developing an IP strategy for long-term market advantage.

The International Patent Classification divides the world of technology into eight sections, which as illustrated below:

SECTION A — HUMAN NECESSITIES

SECTION B — PERFORMING OPERATIONS; TRANSPORTING

SECTION C — CHEMISTRY; METALLURGY

SECTION D — TEXTILES; PAPER

SECTION E — FIXED CONSTRUCTIONS

SECTION F — MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING

SECTION G — PHYSICS

SECTION H — ELECTRICITY

The above mentioned eight sections of the International Patent Classification has over 70,000 subdivisions. Each subdivision has a symbol consisting of Arabic numerals and letters of the Latin alphabet. For e.g., as discussed above, the IPC C12N11/16 relates to “Enzymes or microbial cells being immobilised on or in a biological cell”.

IPC classification symbols are made up of a letter denoting the IPC section (e.g. C), followed by a number (two digits) denoting the IPC class (e.g. C12), then a letter denoting the IPC subclass (e.g. C12N). A number (variable, 1-3 digits) denotes the IPC main group (e.g. C12N11). This is followed by a forward slash “/” and a number (variable, 1-3 digits) denoting the IPC subgroup (e.g. C12N11/16). To elaborate more on IPC classification symbols the following example can be considered:

C: CHEMISTRY; METALLURGY

C12: BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N: MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF

C12N11: Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof

C12N11/16: Enzymes or microbial cells being immobilised on or in a biological cell

C12N11/18: Multi-enzyme systems

Visit our Business Blog homepage: http://research.techcorplegal.com/

International Technology Business & Patent Law Firm in India: Patent Drafting, Patent Searching, Patent Filing in India, PCT National Phase Filings in India: http://www.techcorplegal.com/

International Business & Management Company in Singapore: Starting a Business in Singapore: http://www.techcorpgroup.com/

Contributors: Prity Khastgir andRahul Dev

We regularly update ourFacebook pageand share similar stories onTwitter. You can also check out our premium service offerings for Mobile Applications, Social Media & Cyber LawsandPharmaceuticals, Biotechnology, Food & Healthcare
To know more about us, get connected with us on LinkedInor mail us atinfo [at] techcorplegal [dot] com

Our Business Law Services: Company & Individual Representation, Business Litigation & Corporate Law, Business formation & Review of Business Formation Documents, Cyberlaw, New Media, & Internet/Technology, Entertainment, Sports, & Media, Contract Negotiation, Review, & Drafting, Intellectual Property (“IP”), Real Estate Transactions and Landlord/Tenant Disputes, Employment Law, Securities, Mortgage, Banking & Finance, Mergers & Acquisitions, Legal Malpractice / Ethics Violations / Company Policies / Website Terms & Conditions, Brand Identity & Positioning, Brand and Intellectual Property Licensing and Development, Compliance, Preparation of Business Plans, Business Process, Efficiency Consulting, & Outsourcing, Investment & Strategic Ventures, Project Finance & Management, Competitive Landscape Analysis, Right Time to Incorporate a Legal Entity, Finance, Funding & Venture Capital, Type of Entity: Company vs. Partnership vs. Sole Proprietorship, Right Location (Jurisdiction) to Incorporate a Company, Company Incorporation in India, Partnership, Sole Proprietorship & LLP in India, Setting up Liaison / Representative / Branch/ Project Office in India, Franchising in India, Licensing in India, Retail in India, Things to do Before Quitting a Job and Starting a Business, Selecting a Business Name, Branding & Trademarks, Shareholders & Stock Options, Tax Planning & Management, Employees, Interns, Human Resource (HR) Management & Labor Laws, Joint Ventures, Corporate Restructuring, Mergers & Acquisitions, Commercial Disputes, Litigation & Arbitration, Foreign Direct Investment & Foreign Exchange Regulations in India

Our Patent, Trademark & Legal Services: Patent Searching: Worldwide Patent Searching, Patentability Search, Invalidation Search, Freedom to Operate/ FTO Search, Infringement Search, State of the Art Search, Patent Process and Support Services Outsourcing: Patent Drafting, Patent Illustrations, Patent PCT Filing in India: Patent Filing, Market Research before International Filing, Patent Prosecution and Patent Grant Services in India: Patent Prosecution in India, Preparing Response to Office Action, Post Patent Grant Services for Licensing Inventions: Patent Licensing, Licensability Analysis, Patented Technology Commercialization Services for Individuals, SMEs, Companies: Patent / Technology Commercialization, Due Diligence & SWOT Analysis, Worldwide Trademark/ TM Searching and Filing Services: Trademark Searching, Trademark Filing, Trademark Prosecution & Licensing, Trademark Due Diligence, Legal Research, Process and Support services Outsourcing: Legal Research, Contract Management, Litigation Support, Patent Litigation Support, Medical Litigation Support

So here it is, the end of summer. The beginning of Autumn. I’m leaving the NIH and moving back to Oxford November 1. This is a rather arbitrary date, but then again, as I’ve come to learn, many things in the life of a PhD student are arbitrary. For a long time the date was October 1st, but then a shipment of mice back in May was backordered by several weeks, so it got moved back a month.

November 1. 39 days.

So far, everything seems to be reasonably under control. I learnt how to do qPCR last week so there will be a lot qPCR stuff. And the last big surgery was done on Thursday of last week, with the DIVAA transplants coming out in 2 days. Oh, and my final regular transplants were embedded yesterday and will be sectioned tomorrow. Finally, everything seems to be coming together.

Except for the staining. In order for my grand PhD scheme to work I need to stain and quantify the amount of blood vessels in transplants. So far, I haven’t yet gotten this to work, and it’s imperative that it starts to work soon. Because I intend to retroactively stain all the slides I’ve made during my. This quantity to get through is really large, and will take time to stain, image, quantify, and analyze. The staining is the one huge hurdle to my success. I may have to take some of it to Oxford to finish there, which goes against my plan of just writing and not doing any bench work in Oxford – but hopefully I can finish before then.

So… I’ll keep updating the blog regularly – the next blog post is going to be about how to measure the velocity of cells. Please also check on me on Twitter (@makingbones) – starting October 1st I’ll be tweeting daily on progress in the countdown towards the big move!

¿Alguna vez os habéis preguntado qué sucede cuando se pierde masa muscular debido a un accidente o una enfermedad?. ¿Cómo se regenera, si es que es posible?. No es fácil “crear músculos nuevos” o regenerar musculatura. Hasta ahora, lo más común es el uso de prótesis en determinados casos.

Nuevas investigaciones han logrado regenerar musculatura en pacientes. Estas noticias hacen que se pueda plantear la regeneración muscular en un futuro como opción terapéutica. La clave de la regeneración muscular es la matriz extracelular, que es el tejido que da estructura a órganos y tejidos. Hasta hace muy poco, su única función conocida era precisamente ésa: dar estructura. Sin embargo, nuevos resultados de investigación han revelado que también tiene entre sus funciones aquellas de crecimiento y reparación tisular. En estos experimentos se utilizó matriz extracelular de mamíferos, la más cercana a la humana es la de los cerdos, para regenerar el músculo humano. Esta matriz es capaz de comunicarse con las células adyacentes y estimula la creación de tejido muscular nuevo a la vez que actúa confiriendo estructura al nuevo músculo. Estos resultados, aunque preliminares, hacen pensar que en un futuro podría llegar a utilizarse este método en pacientes afectados con pérdidas severas de masa tisular.

Now we have learned that technology has made it possible one more time. Muscle tissue is able to grow on human patients. The key lies on the extracellular matrix, which is the tissue that gives structure to tissues and organs. Until recently, its only function was thought to be precisely that: give structure. Recent investigations have revealed that it is also responsible for repair and growth functions. With the aid of extracellular matrix from other mammals, the closest to humanswould be pigs, human muscle cells are able to regrow.  The extracellular matrix communicates with the cells of the host and start creating muscle tissue, acting as the scaffold guiding the new cells. These results hold great promise although they are preliminary and many more studies are needed before it translates efficiently into the clinic and can be used as a regular procedure.

This is an excellent video made by the BBC in talks with Professor Post at Maastricht University, which addresses many of the key issues. Briefly, there are some very important issues to bring up.

Macro-impact:
o Growing meat in the current way has huge environmental effects based on the amount of food being fed to animals, and the amount of waste being put out
o Not discussed: the effects on the economy in transitioning from an agricultural system to a biological factory system.
o Also not discussed: what would happen to all the waste from a meat factory?

Taste:
o No one knows exactly why meat tastes the way it does.
o Fat, which is believed to give flavor to meat from an animal, would have to be grown separately and mixed in, and there is no saying that fat from a dish tastes like fat from an animal. Back to square one.
o Texture was not directly discussed in the video, though as it appears that Professor Post is applying mechanical pressure to cells (conjecture based on an apparatus I observed in the video); this would lead us to the conclusion that he is actually producing muscle tissue and not just layers of muscle cells – an important distinction.

Scale
o Professor Post rightly talks about the vascularization issue, which I talked about in my tissue engineering post on August 5 – it’s hard to grow a thick chunk of meat in a dish because of the inside-outside issue.
o Professor Post suggests bioreactor systems, but these still produce many thin layers that would have to be pressed together to form a steak (a bit like a really thick version of chicken slices seen at the deli counter).
o When Professor Post talks about the $250,000 burger, he’s almost certainly going to take many of these smaller pieces and put them together to get the final product
o Scale is also a problem because, although cells can be exponentially expanded in culture, as discussed in my August 11 post on passaging cells their characteristics change with each passage out of the animal. (I presume he is not talking about using immortalized cell lines because he intends to keep donor animals.)

Summary: Professor Post is working on a project that, for obvious reasons, gets a lot of attention from mainstream media. He is obviously well aware about the limitations of his project, but is taking a practical approach to getting it done right. It’s a great theory, and deserves investment, but we shouldn’t expect to be eating faux meat (“in vitro meat”) for years to come.

You can’t just slice willy-nilly and eat the results. I don’t even want to think about eating tissue engineered meat – that’s a blog post for later this week.

One method is indeed to freeze it, that’s called “cryo-sectioning”, “cryo” actually being Greek for “icy cold”! Another technique is to embed the tissue in plastic and then cut it. Ever tried to cut plastic accurately? Only a small handful of technicians in the US are experts in that. But let us think about the reasons why the stir-fry recipe tells you to freeze the meat. As the meat freezes it becomes harder, and more stable; hence, easier to cut.

What we can do is embed the samples in wax. Wax infiltrates the entire tissue (if it’s soft) and stabilizes it, making it possible to cut thin slices without any squishing. And the slices that we can cut are super-thin! A microtome could slice a human hair. Length-wise. This means that we can very precisely see what is going on in the tissue that we have made.

To skip a lot of the science jargon, the process is basically thus:

1. The tissue is slowly transitioned to being in a wax solution.
2. The tissue can then be placed in a mold with more wax and let to cool.
3. The mold is then removed and the block can be cut

Ever seen a block of amber with a fly or something prehistoric in it in a natural history museum? That’s what this is like, except unfortunately the samples don’t look nearly as cool as flies. And they’re in wax, not amber; so aren’t worth much unless you’re the scientist that made them. My precious…….

So there you have it! The non-scientist’s version of how to slice bone.

According to the September issue of Gastroenterology, sutureless, endoscopic transplantation of sheets of autologous oral mucosal epithelial cells safely and effectively promotes re-epithelialization of the esophagus after surgery.

Superficial esophageal neoplasms are often removed by esophageal endoscopic submucosal dissection (ESD). However, this leaves an ulcer that takes 4 weeks or more to heal, and many patients develop esophageal strictures (narrowing or tightening of the esophagus). The strictures cause dysphagia and can require balloon dilation.

Ohki et al. collected interior buccal mucosal tissue (approximately 6 mm in diameter per cell sheet) from 9 patients with superficial esophageal neoplasms. The epithelial cells were isolated and then cultured for 16 days. Sheets of mucosal epithelial cells (23 mm in diameter) were then collected and transferred, using endoscopic forceps, onto the esophageal ulcer, immediately after ESD (see video).

The procedure of esophageal ESD and endoscopic cell sheet transplantation. Cell sheets were transplanted to the site of ulceration using endoscopic forceps and a support membrane. After a 10-minute wait, the deposit of extracellular matrix underneath the cell sheets allowed the rapid adhesion to the ulcer surface without sutures.

The authors observed complete re-epithelialization within a median time of 3.5 weeks—similar to the normal healing time. However, no patients developed dysphagia, strictures, or other complications following the ESD, except for 1 patient who had a full circumferential ulceration that expanded to the esophagogastric junction.

Ohki et al. conclude that sheets of engineered epithelial cells, fabricated ex vivo from autologous oral mucosal epithelium, effectively reconstruct the esophageal luminal surface and prevent stricture in patients following esophageal ESD.

They propose that the transplanted cells could serve as the source of the regenerated epithelia, because transplanted epithelial cells were detected in biopsy samples of the healed tissue. However, growth factors and cytokines secreted by transplanted cell sheets are also likely to promote wound healing.

In an editorial that accompanies the article, Joshua D. Penfield et al. explain that  autologous epithelial cell sheet transplantation is already being used in the field of burn surgery, and that rapid epithelialization can prevent scarring and contraction of the mucosa. They propose several mechanisms that could facilitate rapid epithelialization of the mucosa, including interactions with mesenchymal cells and secretion of cytokines and other growth factors from the transplanted cell sheets.

Long-term studies with more patients are needed to further assess the benefits and risks of this method. Ohki et al. propose that this technique might be developed to treat patients with larger esophageal tumors, or neoplasms that arise in patients with Barrett’s esophagus.

Read the article online.
Ohki T, Yamato M, Ota M, et al. Prevention of esophageal stricture after endoscopic submucosal dissection using tissue-engineered cell sheets. Gastroenterology 2012;143: 582−588.e2.

Read the accompanying editorial.
Penfield JD, Gorospe EC, Wang KK. Tissue-engineered cell sheets for stricture prevention: a new connection between endoscopy and regenerative medicine. Gastroenterology 2012;143:526−529.

Harvard scientists have created a type of “cyborg” tissue for the first time by embedding a three-dimensional network of functional, biocompatible, nanoscale wires into engineered human tissues.

As described in a paper published Aug. 26 in the journal Nature Materials, a research team led by Charles M. Lieber, the Mark Hyman Jr. Professor of Chemistry at Harvard, and Daniel Kohane, a Harvard Medical School professor in the Department of Anesthesia at Children’s Hospital Boston, developed a system for creating nanoscale “scaffolds” that can be seeded with cells that grow into tissue.

“The current methods we have for monitoring or interacting with living systems are limited,” said Lieber. “We can use electrodes to measure activity in cells or tissue, but that damages them. With this technology, for the first time, we can work at the same scale as the unit of biological system without interrupting it. Ultimately, this is about merging tissue with electronics in a way that it becomes difficult to determine where the tissue ends and the electronics begin.”

Contributing to the work were Robert Langer, from the Koch Institute at the Massachusetts Institute of Technology, and Zhigang Suo, the Allen E. and Marilyn M. Puckett Professor of Mechanics and Materials at Harvard’s School of Engineering and Applied Sciences.

The research addresses a concern that has long been associated with work on bioengineered tissue: how to create systems capable of sensing chemical or electrical changes in the tissue after it has been grown and implanted. The system might also represent a solution to researchers’ struggles in developing methods to directly stimulate engineered tissues and measure cellular reactions.

“In the body, the autonomic nervous system keeps track of pH, chemistry, oxygen, and other factors, and triggers responses as needed,” Kohane said. “We need to be able to mimic the kind of intrinsic feedback loops the body has evolved in order to maintain fine control at the cellular and tissue level.”

Using the autonomic nervous system as inspiration, Bozhi Tian, a former doctoral student under Lieber and a former postdoctoral fellow in the Kohane and Langer labs, joined with Lieber and colleagues to build meshlike networks of nanoscale silicon wires.

The process of building the networks, Lieber said, is similar to that used to etch microchips.

Beginning with a two-dimensional substrate, researchers laid out a mesh of organic polymer around nanoscale wires, which serve as the critical sensing elements. Nanoscale electrodes, which connect the nanowire elements, were then built within the mesh to enable nanowire transistors to measure the activity in cells without damaging them. Once completed, the substrate was dissolved, leaving researchers with a netlike sponge, or a mesh, that can be folded or rolled into a host of three-dimensional shapes.

Once complete, the networks were porous enough to allow the team to seed them with cells and encourage those cells to grow in 3-D cultures.

“Previous efforts to create bioengineered sensing networks have focused on two-dimensional layouts, where culture cells grow on top of electronic components, or on conformal layouts, where probes are placed on tissue surfaces,” said Tian. “It is desirable to have an accurate picture of cellular behavior within the 3-D structure of a tissue, and it is also important to have nanoscale probes to avoid disruption of either cellular or tissue architecture.”

Using heart and nerve cells, the team successfully engineered tissues containing embedded nanoscale networks without affecting the cells’ viability or activity. Using the embedded devices, the researchers were then able to detect electrical signals generated by cells deep within the tissue, and to measure changes in those signals in response to cardio- or neuro-stimulating drugs.

They were also able to construct bioengineered blood vessels, and used the embedded technology to measure pH changes — as would be seen in response to inflammation, ischemia, and other biochemical or cellular environments — both inside and outside the vessels.

Though a number of potential applications exist for the technology, the most near-term use, Lieber said, may come from the pharmaceutical industry, where researchers could use it to more precisely study how newly developed drugs act in three-dimensional tissues, rather than thin layers of cultured cells. The system might also one day be used to monitor changes inside the body and react accordingly, whether through electrical stimulation or the release of a drug.

The study was supported by the National Institutes of Health, the McKnight Foundation, and Children’s Hospital Boston.

http://news.harvard.edu/gazette/story/2012/08/merging-the-biological-electronic/

Related articles

• Merging bioengineering and electronics: Scientists grow artificial tissues with embedded nanoscale sensors (phys.org)
• Nanoscale scaffolds and stem cells show promise in cartilage repair (nextbigfuture.com)
• Tissue Engineering the Brain, Eyes and Pituitary Glands (nextbigfuture.com)

Why do we freeze cells? Logistically, we freeze cells because we get our cells from living specimens who donate valuable cells and aren’t infinite sources. Also, although I pointed out how cells expand exponentially in my post on August 11th, they only do this near the beginning of their lives and undergo a process known as “senescence” at later age when they stop growing, unless they are true stem cells. Additionally, cell behavior and differentiation have been shown to change with continued expansion in a Petri dish, until cells might not be a good representation of their origins. Therefore, if we get a higher number of cells that we need at low passage (see the August 11th entry), we freeze them. For example, in our lab we never use our bone marrow stromal cells above passage 5 for transplants, and above passage 10 for any studies at all.

Mouse and human body temperatures are 37°C, and therefore, that’s what the incubator temperatures in our lab are always set at, to allow cells to grow at a normal rate, without causing them undue stress. As cells get colder, all of their biological processes slow down, but importantly, do not die (or proliferate). Therefore, freezing provides an excellent way of keeping cells exactly the way they are for long periods of time.

“But”, I hear you say, “surely the ice crystals that form during freezing will puncture and kill all of the cells?” That’s exactly the reason why we can’t freeze cells in normal growth medium (liquid) but instead using “freezing medium”. This typically contains 10% DMSO (dimethyl sulfoxide) which actually helps protect cells from rupturing, and is known as a “cryoprotectant”. However, we can’t use just DMSO because it is actually toxic to the cells. Therefore, the rest of the solution is generally made up of components that one would find in normal growth medium (e.g. FBS [foetal bovine serum]). There are also many different proprietary freezing mediums on the market.
The Procedure:
1) Make, or buy, freezing medium: I use 10% DMSO, 90% FBS
2) Trypsinize and centrifuge cells as if you were passaging them
3) After removing the media/trypsin solution, resuspend the cells to 1×106/mL (or as desired, but not more than 4×106/mL) in freezing medium
4) Portion the cell solution into freezing vials, 1mL per vial
5) Place vials in an isopropanol chamber (a special freezing apparatus that slows down the freezing process to avoid shocking and killing cells) and place container in a -80˚C freezer
6) The next day, transfer to a normal container box, and, if desired, transfer the cells in liquid nitrogens

Dr. Sasai has invested a great deal of time and dedication to his dear project and his hard work has paid off. He has been able to grow several layers of the cerebral cortex and a rudimentary pituitary gland. His next challenge is to build a cerebellum, which is responsible for movements and equilibrium. By accomplishing this great feat, he will be able to study how brain parts work and shed light on unanswered questions in the field of neurology.

But what has Dr. Sasai done differently than other researchers and what are the keys to his success?. For decades, teams of investigators have tried very hard to find the key to tissue differentiation, which would open the door to growing organs in the laboratory. Well, he armed himself with patience, dedication, and passion and worked tirelessly in the lab to recreate a controlled environment with the right chemical signals to recreate what really happens in an embryo during gestation, where stem embryonic cells differentiate themselves into different types.

The primarily, and long-term, application of this news are obviously directed at facilitating certain types of transplants, as well as the ability to study the structure and function in detail of these organs and tissues. One good example of it, and the one Dr. Sasai and colleagues are currently working on, is to implant lab-grown retinas in animal models.

Science Daily, 2008: First Tissue-Engineered Trachea Successfully Transplanted

In this article we see that the scientists utilized two of the three components I talked about in my August 8th post, i.e. cells and scaffold. The scaffold, in this case, is a 7cm section of decellularized trachea from a donor. The donor was deceased – it is not possible to donate a trachea section while still alive. 7cm is a really large area to consider in terms of modern tissue engineering, as much research is done on a much smaller scale. The term decellularized means that the tissue was stripped of all cells to make it less immunogenic to the recipient. However, the physical structure of the organ is retained and, importantly, its biological activity, as the proteins that make up the structure are kept and still active. This scaffold was then seeded with cells that had been collected from the recipient, i.e. autologous cells, so that there would be no graft/host response. Seeding the cells is important because it provides the organ with a dynamic, living presence instead of just implanting an empty construct.

New York Times 2011: Synthetic Windpipe is Used to Replace Cancerous One

In this article we see that a similar approach of seeding autologous cells on a tracheal scaffold was taken. However, in this instance the scaffold material is different, instead of being a donated natural scaffold, it is a synthetic polymer material. This has several associated pros and cons. The pros of using this approach are that it doesn’t require a donor and associated organ processing logistics, but, more importantly, the scaffold can be shaped to fit the recipient exactly using modeling and advanced fabrication techniques, whereas donor tracheas are often not a good fit. The major con of the system is that the scaffold is inert and not biologically active and covered in normal tracheal proteins the way that the previous scientific group described. In order to overcome this issue, the group at the Karolinska Institute also grew the cells on the scaffold in a bioreactor, but added in several growth factors that they hoped would induce the cells to differentiate as desired, and start the biological pathways that would continue to affect how the cells behave even after transplanted.