CA2685964A1 - Methods of fabrication and vascularization of thick three dimensional tissue constructs - Google Patents
Methods of fabrication and vascularization of thick three dimensional tissue constructs Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The present invention describes processes and methods of fabricating and vascularising thick, three-dimensional, tissue constructs by preparing the constructs or portions thereof for freezing or vitrification with cryoprotectant solutions and/or including one or more cryoprotectant solutions as manufacturing materials in computer aided design, manufacturing, assembly and printing systems, (also known as bio-printing or organ-printing systems) and dispensing the prepared cells (or prepared self assembling cell aggregates) into a desired shape along with other desired materials and vitrifying or freezing the tissue construct which can be stored in a vitrified or frozen state by transporting the construct into a holding vessel (such as a vat) where more constructs of a similar nature can be added to the vat and may in some instances create a larger more elaborate structure such as a human organ which can than be removed from a vitrified or frozen state by warming the vat or transferring the construct to a holding vessel which will be equipped for allowing cellular adhesion to occur between the different constructs and self assembling tissue spheroids that have not yet had a chance to assembly due to their frozen or vitrified states. Also discussed are a variety of means for perfusing or vascularizing the construct comprising attaching the construct to a naturally existing or fabricated circulatory system comprising systems and methods of perfusing and circulating blood, nutrients, growth factors and other required materials through the construct.
Description
Industry industrle 41hq/J 'rihwD
Canada Canada IILIIiIIiIIIIIII~IIIIIIIIIIiIIIIIIIIIII~IIIIIIIILIIIIIilllill II 041- 11 CPO oPiC 19880550 Patent Application of John Archie Gillis for TITLE: METHODS OF PRESERVATION, FABRICATION AND PERFUSION OF
TISSUE CONSTRUCTS
APPLICATION NUMBER 2,685,964 CROSS-REFERENCE TO RELATED APPLICATIONS Not Applicable FEDERALLY SPONSORED RESEARCH Not Applicable SEQUENCE LISTING OR PROGRAM Not Applicable BACKGROUND OF THE INVENTION-FIELD OF INVENTION
The present invention relates to methods of fabrication, preservation, banking, transporting, vascularization and perfusion of tissue constructs.
BACKGROUND OF THE INVENTION-PRIOR ART
Tissue engineering in its early days was considered a sub-field of biomaterials. It has recently grown in both importance and potential and is now considered to be a field of its own.
It generally uses a combination of cells, engineering, materials methods, and suitable biochemical and physio-chemical factors to improve or replace biological functions. Tissue engineering is usually describes as an interdisciplinary field incorporating elements of engineering, material and life sciences.
Most recently tissue engineering has begun to incorporate elements of computer aided design and rapid prototyping. The names currently most in use are bioprinting and organ printing.
Tissues are often fabricated in the laboratory using stem cells, growth and differentiation factors, biomaterials, printing devices and biomimetic environments. It is with these combinations of engineered extracellular matrices (or scaffolds), cells, and, biologically active molecules that researchers in this field have propelled this area of research forward.
One of the major challenges facing tissue engineering today is the requirement for more complex functionality. For a greater number of tissue engineered structures to be considered useful in areas such as transplantation, more biomechanical stability is required along with an advanced means of supplying these structures with nutrients, especially when discussing thick tissue structures.
A cryoprotectant is a substance that is used to protect biological tissue from freezing damage. This damage often occurs due to the formation of ice. Cryoprotectants in common use include glycols, such as ethylene glycol, propylene glycol and glycerol and dimethyl sulfoxide (DMSO), 2-methyl-2, 4-pentanediol (MDP) Sucrose and Trebalose.
Cryobiologists have been using both glycerol and dimethyl sulfoxide for decades to reduce ice formation in sperm and embryos that are cold-preserved in liquid nitrogen.
Mixtures of cryoprotectants have less toxicity and are more effective than single-agent cryoprotectants. A mixture of formaxnide with DMSO, propylene glycol and a colloid, was for many years the most effective of all artificially created cryoprotectants.
Cryoprotectant mixtures have been used for vitrification, i.e. solidification without any crystal ice formation.
Vitrification has important application in preserving embryos, biological tissues and organs for transplant. Vitrification is also used in cryonics in an effort to eliminate freezing damage.
Some cryoprotectants function, by lowering a solution's or a material's glass transition temperature. In this way, the cryprotectants prevent actual freezing, and the solution maintains some flexibility in a glassy phase.
Vitrification techniques utilize low toxicity solutions and optimized cooling and warming curves that, when applied under sterile conditions, allow for better, longer, safer and more convenient storage of complex living systems.
An example of a method of cryopreservation of tissues by vitrification is Khirabadi; Bijan S., Song; Ying C., Brockbank; Kelvin G. M. "Method of cryopreservation of tissues by vitrification", Organ Recovery Systems, Inc. US 7,157,222, (2007) or US
6,740,484 This prior art teaches a method that includes vascularized tissues and avascular tissues, or organs. The method comprises immersing the tissue or organ. in increasing concentrations of cryoprotectant to a cryoprotectant concentration sufficient for vitrification;
rapidly cooling the tissue or organ to a temperature between -80° C. and the glass transition temperature (Tg); and further cooling the tissue or organ from a temperature above the glass transition temperature to a temperature below the glass transition temperature to vitrify the tissue or organ.
This prior art also describes a method for removing a tissue or organ, from vitrification in a cryoprotectant solution. The method comprises slowly warning a vitrified tissue or organ in the cryoprotectant solution to a temperature between -80° C. and the glass transition temperature; rapidly warming the tissue or organ in the cryoprotectant solution to a temperature above -75° C.; and reducing the concentration of the cryoprotectant by immersing the tissue or organ in decreasing concentrations of cryoprotectant.
With this method for treating tissues or organs, viability is retained at a high level. For example, for blood vessels, the invention provides that smooth muscle functions and graft patency rate are maintained.
These and similar methods are great for protecting existing and fabricated tissues from damage, but are not always successful at penetrating deep into thick tissue constructs. These methods have not been used in tissue engineering processes such as those described by the present invention. It is an object of the present invention to prepare cellular compositions with both intracellular and extracellular cryoprotectant solution mixtures prior to a bio printing process, thus allowing precise placement of solutions. In fact cryoprotectants are rarely if ever used in tissue engineering. Most cryoprotectants have been used in protecting existing structures. It can be very difficult to position the protective solutions deep within these already existing structures. This ability of the protective solutions to be selectively located is one of the key benefits of the described invention.
Preservation of organs and tissues are commonplace in medicine, but because organs are most often donated rather that fabricated it can be difficult to place these solutions in areas that can deeply penetrate the structure, especially if the tissue or organ is a thick structure.
Organ printing is usually assisted by computers, dispenser-based, and has an emphasis on three-dimensional fabrication. These methods are aimed at constructing functional organ modules however at present there has been limited success and the printing of entire organs layer-by-layer has not yet been. realized.
Bio-printing or organ printing is a new area of research and engineering that involves printing devices that deposit biological material. Examples of bioprinter technologies would be those in development by Organovo and fabricated at Inventech, which use combinations of "bio-ink" and "bio-paper" to print complex 3D structures.
A number of developments have been occurring in the field of organ printing.
One such development is that of Self-Assembling Cell Aggregates. Forgacs; Gabor;
(Columbia, MO) ;
Jakab; Karoly; (Columbia, MO) ; Neagu; Adrian; (Columbia, MO) ; Mironov;
Vladimir; (Mount Pleasant, SC)"Self-Assembling Cell Aggregates and Methods of Making Engineered Tissue Using the Same", The Curators of the Univeristy of Missouri, Columbia MO, US20080070304,2008 This prior art describes a composition comprising a plurality of cell aggregates for use in the production of engineered organotypic tissue by organ printing. In a method of organ printing, a plurality of cell aggregates are embedded in a polymeric or gel matrix and allowed to fuse to form a desired three-dimensional tissue structure. An intermediate product comprises at least one layer of matrix and a plurality of cell aggregates embedded therein in a predetermined pattern. Modeling methods predict the structural evolution of fusing cell aggregates for combinations of cell type, matrix, and embedding patterns to enable selection of organ printing processes parameters for use in producing an. engineered tissue having a desired three-dimensional structure.
Another development is the method of forming an array of viable cells developed by James Yoo, Tao Xu and Anthony Atala which decribes a method wherein at least two different types of viable mammalian cells are printed on to a substrate. Inventors:
James Yoo, Tao Xu, Anthony Atala. Application number: 12/293,490 Publication number: US
A I Filing date: Apr 20, 2007 These methods of tissue engineering still suffers from some of the limitations of traditional scaffolding methods. There have been some great successes with this method, but the issue of nutrient delivery is still a major, concern.
A common problem with thick tissue structures is that cells deep inside the structure are damaged due to a lack of nutrient delivery. One can delay this problem for a short by preserving the tissue with. a cryoprotectant solution, but unless the tissue is prepared as described in the present invention the problems of getting cryoprotectant solutions into all the desired locations, including cells deep within the structure remains a large and limiting problem.
If tissue engineering is ever to surpass the tissue thickness limit of 100-200 m, it must overcome the challenge of creating functional blood vessels to supply cells with oxygen and nutrients and to remove waste products.
A major dilemma with most current tissue engineering technologies is that most tissues and organs require vascularization and perfusion to survive. Creating this vascular supply and more viable methods of perfusion to a thick-engineered tissue construct remains one of the great challenges in the field today.
SUMMARY
By immersing cells and cellular aggregates, in gradually increasing cryoprotectatnt solutions prior to their dispensing from a three dimensional printing technology we can create a structure that is very well prepared for preservation.
Once cooled the tissues can be transported or banked for drug testing, cell therapies, graphs and implantation, reconstructive surgery, wound healing, cardiovascular treatment and many others beneficial applications.
When the constructs are taken out of their preserved state they will be moved from their vat and into a new holding vessel. The holding vessel will have holes for transporting substances; will contain bioreactor and perfusion bioreactor components, a temperature specific environment and electronic pin molding capabilities.
The tissue selection located in, the holding vessel will then be attached to a circulatory system of a human by connecting the vasculature of the human to an umbilical cord. The other and of the umbilical will be attached to the vasculature of the tissue selection. A tube casing containing a protective solution will protect the cord.
BREIF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a flow chart describing how one or more cells may be prepared for bio-printing and dispensed as a cellular composition that is ready for cryopreservation.
Fig. 2 is a flow chart describing how a number of tissue constructs prepared for preservation can be placed in a vat, preserved, stored and/or transported and warmed.
Fig. 3 shows what could be separate sections of a printed heart valve.
Fig. 4 shows what the separate sections of a printed heart valve could look like if stacked together in a holding vessel.
Fig. 5 shows what the separate sections could look like once fully self assembled into a finished structure.
Fig. 6 shows what a pin molding system capable of adjusting to different shapes for compensating tissue compaction, maturation and movement could look like. The mold would also be helpful with supporting materials involved in extracellwar matrix maturation.
Fig. 7 shows a human being perfusing a. tissue structure or organ by means of attachment to his/her circulatory system. The holding vessel is a Transm.edic device with the organ enclosed inside and attached to the human via an umbilical cable donated from a new born child and enclosed in a protective tube.
DRAWINGS-Reference Numerals - Cryoprotectant Solution 12 - One or more cells 14 - Preparation of cells for preservation 16 Bio-paper 18 - Cryo-prepared cells assembled into self assembling tissue spheroids/bio-ink - Other materials 22 - Dispensing system 24 - Output from dispensing system containing spheroid shaped cryo-prepared cellular compositions situated for the process of self assembly 26 - Tissue Construct #1 28 - Tissue Construct #2 - Tissue Construct #3 32 - Vat 34 - Means of cooling a tissue selection 36 - Means of storage and transport 38 - Means of warming a tissue selection 40 - Means of transferring a tissue selection into a molding system 42 - Section. or layer of a tissue selection to be assembled into a larger structure.
44 - Large tissue structure fabricated from smaller portions 46 - One pin of a pin mold 48 - Pin mold pin holding portion 50 - Protrective holding vessel for tissue selection(s) 52 - Holes for nutrient, blood and supply delivery 54 - Vasculature 56 - Organism with circulatory system that will. supply nutrient delivery and waste removal to a tissue selection.
58 - Transmedic style holding vessel 60 - Protective tube that holds umbilical cord DETAILED DESCRIPTION OF THE DRAWINGS
Fig. 1 is a flow chart showing cryoprotectant solutions 1.0 and one or more cells 12 coming together wherein they are provided with a means of being prepared for preservation 14. The prepared cells of 14 are assembled into self-assembling tissue spheroids or what is known in the art as bio-ink IS. Loaded into a dispensing system 22 are the bio-ink 18, the bio-paper 16 and other materials 20 which may include other cryoprotectant solution, matrix materials, scaffolds and gels. From the dispensing system 22 we get an output containing spheroid shaped cryo-prepared cellular compositions situated for the process of self-assembly 24 into a desired shape, pattern or three dimensional structure.
Fig. 2 is a flow chart showing a number of different tissues 26, 27, 28, which will be loaded into a vat 32 with a shape complementary to the shape of the printed tissue selections.
A means of cooling 34 will be provided and when cooled to a desired temperature the tissues will be stored and/or transported 36. When the tissues reach their location or it is desired to remove them from their cryopreserved state a means of warming 3 8 will be provided so as to enable transfer to a pin molding system 40 or for other uses.
Fig. 3 is a diagram of a heart valve printed in layers or sections 42. Each section 42 was printed as a separate unit with a specific shape. At this stage of the process we can see how when the layers are placed together that they will form. the shape of a heart valve.
Fig. 4 is a diagram of our layers 42 stacked together to form a structure 44 that will be coaxed into self assembly and form the shape of a heart valve.
Fig. 5 shows what separate sections of a heart valve could look like once fully assembled into a finished structure 46.
Fig. 6 provides a visual example of what a pin molding system capable of adjusting to different shapes for compensating tissue compaction, maturation and movement could look like. 42 shows a holding vessel that has a means to provide a protective environment for the tissue selections. In the preferred embodiment it will be a membrane like encasing made from a material that is flexible, malleable and capable of a variety of shapes so that when self assembly, fusion, maturation, compaction or change in shape occurs to the construct our pins 46 can move to compensate for these changes and in most instances prevent unwanted areas of the structure from moving into undesirable locations. 48 is a stand for holding the pins of our mold. 46 shows pins that when moved together can create a desired shape or mold that can change over time for allowing the same casing or holding vessel the ability to provide structural support during changes to the structure. Holes 52 are provided to the holing vessel to allow transport of materials to and from the tissue selection(s) from an outside source.
Vasculature 54 is shown passing through the holes. The vertical three-dimensional image screen described in patent number: 4654989 Filing date: Aug 16, 1.985 Issue date: Apr 7, 1987 descibes a screen with ability to mold to many objects. United States Patent 6,625,088 to Mah; Pat Y. and Tinier; Robert Bruce issued on September 23, 2003 describes a similar pin display device that has an electronic mechanisms for moving its pins into different shapes.
Fig. 7 shows a human being or patient 56 perfusing a tissue structure or organ by means of attachment to their circulatory system. The holding vessel is a Transmedic device 58 with the organ enclosed inside and attached to the human via an u#iobilical cable donated from a new born child and enclosed in a protective tube 60.
DETAILED DESCRIPTION OF PREFERRED EMBODNTS
Reference will now be made in detail to various embodiments of the invention, one or more examples of which are set forth below. Each embodiment is provided by way of explanation of the invention, not limitation, of the invention. In, fact, it will be apparent to those skilled in the art that various modifications and variations may be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, may b used in another embodiment to yield a still further embodiment. Thus, it is intended that the present invention cover such modifications and variations as come within the scope of line appended claims and their equivalents.
In the preferred embodiments the present invention describes a number of steps for the fabrication, preservation and perfusion of a tissue constru t.
The process begins by immersing cells in varying level) s of cryoprotectant solutions. The cells are then aggregated into self-assembling tissue spheroids, and dispensed into a desired shape.
The innovative method comprises ink jet printing a cei composition onto a substrate wherein the cells within the composition have been prepar~d for cryopreservation, cooling, freezing or vitrification. A great example of Ink jet printing of viable cells is US Patent 7,051,654 Boland; Thomas (Suwanee, GA), Wilson, Jr.; illiam Crisp (Easley, SC), Xu; Tao (Clemson, SC), which, is hereby incorporated by reference in its entirety. It describes a method for forming an array of viable cells. In one embodi' cnt, the method comprises ink jet printing a cellular composition containing cells onto a substrate. Upon printing, at least about 25% of the cells remain viable after incubation for 24 hour at 37° C.
in a 5%
CO2/95% O2 environment.
In the preferred embodiment the cultured cells that are included in the cellular composition to be printed are prepared with. varying levels of cryoprotectant solutions. A
variety of solutions can be used to generate various levels of results and successes.
Examples of some potential methods that may be used in whole or in part include, but are not limited to "Method of cryopreservation of tissues by vitrification" (Khirabadi; Bijan S., Song;
Ying C., Brockbank; Kelvin G. M. "Method of cryopreservation of tissues by vitrification.", Organ Recovery Systems, Inc. US 7,157,222, 2007), The cryogenically prepared cells will form a. bio ink that will be loaded into a three dimensional fabrication device. A great example of a bio ink is US Patent Application 20080070304 to Forgacs; Gabor; (Columbia, MO) ; Jakab; Karoly; (Columbia, MO) ; Neagu;
Adrian; (Columbia, MO) ; Mironov; Vladimir; (Mount Pleasant, SC) "Self Assembling Cell, Aggregates and. Methods of Making Engineered Tissue Using the Same", which is hereby incorporated by reference in its entirety and explains bio ink and bio paper.
No prior art reference provides a description of a process incorporating the use of cryogenic preparation of cells or cell aggregates for the purpose of being loaded into a printer.
This is one of the novel features of the present invention. With prior methods of applying cryoprotectant solutions to some tissue constructs, (especially into think constructs or organs) it has been found difficult if not impossible to get the cryoprotectant solutions to the desired locations. The present invention provides a remedy for this problem.
After being dispensed from an ink jet printer the cellular spheroids or aggregates will be preserved by methods of freezing or vitrification. The construct will be stored and transported for cell therapies, drug tests, and in the preferred embodiment oftbe present invention used, as a section to be fused with other similar sections to create a larger construct.
The prepared cells are then placed into a vat or similar cooling device, next to one or more other cellular constructs that have been prepared in a similar manner. The constructs are then.
vitrified or frozen. The cells may now be banked or transported for drug testing or cell therapies and once taken out of their preserved state they will be coaxed into self=assembly and fused together to create a larger structure.
One of the reasons the present inventor feels it is pertinent to create and preserve the tissues in sections is so that they can be easily stored at a later time when they are needed, and so that the bioprinting system is freed up for use. Another reason for printing the constructs in.
sections is that some bioprinters will be designed and/or set up for the creation of specific tissue structures. As an example; bioprinter I would be programmed to print section A of a kidney with specific materials and preservation solutions and thus the technician is trained to know exactly what is required each time, while bioprinter 2 is programmed to print section B
of the same kidney with their different but specific materials and preservation solutions. The process continues with bioprinter 3 doing section C and so on., until all the required parts are constructed. By printing the sections simultaneously we can decrease the time it takes to complete the more elaborate structure.
In the preferred embodiment the printers will be programmed to complete the sections at the same time so that they can be placed into a vessel for preservation. Once they are preserved they can be transported to the geographic location in which they are needed.
A viable method of transportation is also a. very beneficial, outcome of the present invention. Organs and tissue assays do not last long if not protected properly.
Bioprinting labs are very expensive and require expertise in operation. As an.
example Inventec sells their bioprinters for $250,000.00 a pretty steep price for any lab. These labs and expertise are only located in a few geographic locations, but with the described preservation methods, preserved tissues fabricated with computer and roboticially targeted.
precision will make it much more viable for the transportation of these products for their use in cell therapy products, tissues, organs and tissue engineered constructs.
The cryogenically prepared cells are printed in layers, and as the layers are completed they are mechanically lowered into a vat, and put into a vitrified or frozen state.
The layers are organized so that they fit together in a desired shape or pattern that will allow the proper portions to fuse in the correct areas when taken out of their vitrified or frozen states. An example of this is to position a vascular network such that when cellular adhesion (self assembly) occurs it will become one unit and thus when taken out of a vitrified or frozen state the sections will fuse together in the correct and desired locations, and be ready to be placed. in a device capable of providing the required supply of nutrients and materials.
Vasculature has been bioprinted in the labs of Anthony Atala without cryopresevation included to a limited degree. It has also been successfully attached to a perfused bioreactor.
In a preferred embodiment self-assembly may occur after preservation, however in alternative embodiments it will occur prior to preservation.
When the individual sections are placed into a vat, the vat will be shaped as to support the dimensions of the larger structure to be fabricated or preserved. At this point the structures will have been persevered or will undergo a preservation process. The construct is now transported to its required location.
When the structure reaches its destination it is taken out of its state of preservation. At this point the structure can be carefully removed from its vat into a holding vessel with electronically programmable and movable pin molding capabilities for supporting the structures and for also providing support for a protective membrane that will encase the structure in a protective biological environment. This type of protective pin molding biological environment will provide support for the structure yet allow for changes to occur during post processing fusion, retraction, remodeling and compaction. Another object of the holding vessel is its temperature specific environment.
When the structures are taken out of their vitrified state they will be coaxed into self assembly as is described in US Patent Application 20080070304. The self assembling tissue spheroids of each section may have aggregated into a larger tissue structure prior to preservation and thus these fused tissue sections will aggregate to form yet an even larger structure. In another embodiment the smaller sections will also require time for self assembly.
There are a number of instances where the present methods of preservation will be very useful. if the tissue constructs are not needed for some time after printing or if they are required to be transported the present methods will assist with preventing damage and cell death from occurring. Often tissues are transported long distances for drug testing, cell therapies and if there are no bio-printing laboratories near an area where bioprinted structures are wanted or needed, the aggregation methods explained herein may be a very necessary requirement.
Another reason that the described method is practical is the high cost and low'success rate of many other alternatives. Time is of the essence when printing tissue constructs, as they can only be maintained for relatively short periods of time after, printing, before damage occurs.
The present sectioning method can be used without the use of cryogenic solutions integrated into the construction process, but only if a company has a number of bioprinters printing sections of a predetermined structure at the same time. This type of lab would then need to stack the sections into a holding vessel. and perfuse them with nutrients as soon as possible. This method is not practical if the construct is to be transported.
The structures will be provided with nutrients and waste removal using standard methods found in the art, such as bio reactors, perfused bioreactors or solutions used in systems for ex vivo care at new physiologic conditions, however once a vascular structure or vascular system, such as a bioprinted intraorgan branched vascular system has been assembled and becomes mature and functional enough for initiation of intravascular perfusion it will be attached to an umbilical cable. The umbilical cable may be fabricated from human cells or may be one donated by a suitably matched new born baby and then attached to a human circulatory system. This would likely be the circulatory system of the future recipient of the structure.
A group from South Carolina as well as a group led by Gabor Forgacs' have recently demonstrated that building a branching intraorgan vascular tree is a realistic and achievable goal. This issue was also addressed by Peter Wu (University of Oregon, USA) who presented applications of LAB in fabricating branch/stem, structures with human endothelial cells and T
Boland who presented results on thermal inkjet printing ofbiomaterials and cells for capillary constructs. (Cui X and Boland T 2009 Human microvasculature fabrication using thermal ink jet printing technology Biomateri.als 30 6221-7) When the structure has fused into a single unit it will remain in its holding vessel where it will continue to receive nutrients and blood from the human circulatory system. This system.
will also provide the structure with the ability to remove waste.
One of the great benefits of the structure being located outside of the body is that it may be tended to by doctors, engineers and other professionals for other additional procedures, tests or substance delivery that may be beneficial to the survival and maintenance of the structure.
The structure may when required also receive external, electrical stimuli.
Other great benefits of the structure being perfused by the patient's own circulatory system, yet essentially being located outside the body is that it can be accessed, repaired, manipulated and supplied with additional substances or therapies.
Current methods of perfusing a tissue structure are limited, due to time constraints. This is seen in cases of organ donation. When a donated organ is matched with a recipient, it is imperative that the organ reaches the recipient in as short of time as possible. Even with our advanced technologies, helicopters and database matching systems organs are often lost, due to a variety of reasons that include injuries during brain-death, ischemia, cell death and other causes.
Currently there are a number of systems that are perfusing organs such as Transm.edics, "Organ Care System", Organ Recovery Systems "LifePort" technologies and the Toronto XVIVO Lung Perfusion System. The Lung Perfusion Systern is being worked on by Dr. Shaf Keshavjee in the Lung Transplant Program at Toronto General Hospital (TGIF).
They have developed an "ex vivo" or outside the body technique capable of continuously perfusing or pumping a bloodless solution containing oxygen, proteins and nutrients into injured donor lungs. This technique allows the surgeons the opportunity to assess and treat injured donor lungs, while they are outside the body, to make them suitable for transplantation.
These methods of perfusion are great advances in medical technologies, but still have their limitations. The present invention describes that at first seems odd, but is actually the most natural method of perfusing either a transplanted organ or a tissue engineered construct. If we think of how a fetus is perfused in the womb we have a fetus attached to an umbilical cord, which, is attached to its mother. Both the fetus and the umbilical are in a protective solution.
In the present invention we create something very similar. Our fetus is out tissue engineered construct and our mother is the person who will be having the construct or organ implanted into them. In the preferred embodiment the ex vivo perfusion module will be attached via existing or fabricated umbilical cables to the construct or organ to be perfused. The construct or organ will be located outside of the body and housed in a protective temperature specific environment, likely at 37 degrees C and may include a protective solution for, surrounding the construct/organ. The tube attaching to the recipients circulatory system via an umbilical cable will be housed in a tube containing a protective solution, which may contain Wharton's Jelly or a suitable substitute, nutrient composition, or liquid that may assist in sustaining the cord during perfusion of the construct. Connection of this cord will require surgical attachment.
In placental mammals, the umbilical cord (also called the birth cord or funiculus uznbilicalis) is the connecting cord from the developing embryo or fetus to the placenta.
During prenatal development, the umbilical cord comes from the same zygote as the fetus and (in humans) normally contains two arteries (the umbilical arteries) and one vein (the umbilical vein), buried within Wharton's jelly. The umbilical vein supplies the fetus with oxygenated, nutrient-rich blood from the placenta. Conversely, the umbilical arteries return the deoxygenated, nutrient-depleted blood.
Successful perfusion of an extra organ using a similar procedure in vivo has been accomplished in the art by what is known as hetcrotopic surgery. In this medical procedure the patient's own heart is not removed before implanting a donor heart. The donor heart is 1.7 positioned so that the chambers and blood vessels of both hearts can be connected to form what is effectively a'double heart'.
Another example of in vivo perfusion of an extra organ is that of a kidney transplant. In many kidney transplants the original but likely damaged kidneys are left in the recipient.
An example of ex vivo perfusion is that of babies who are occasionally born with organs outside their body and often survive this way for many months prior to having the organs transferred inside their body.
Where the present invention differs from these procedures is that the heterotropic procedure takes place inside an. organism not via an, ex vivo attachment.
Another difference is that in the present invention the organism that the construct or organ is first attached to after being printed acts as a temporary lobby area or location and once the organ has been matured it will be attached to the recipient The tissue constructs of the present invention include portions of, or whole tissues (i.e., bone, cartilage, blood vessels, bladder, etc.) The tissue harvested may consist of any biological material and may include materials that have been manipulated and/or changed from their original state, such as geneticially altered materials or stem cell cultivations.
The dispensing systems of the present invention include computer aided design, manufacturing, assembly and/or printing systems. These systems make use of computer technology to aid in the design, manufacturir, assembly and/or printing of a product.
Examples of such systems include, Direct Digital Manufacturing, Rapid Prototyping, Three Dimensional Printing, Bio-printing, (CAD/CAM), Stereolithography, Solid Freeform Fabrication, Self-Replicating Machines, 3D Microfabrication, Digital Fabrication and Desktop Manufacturing Systems, and the methods and technologies involved, developed and understood by those skilled in the art.
The Bio-printing systems of the present invention will include the use of what is known in the art as bio-paper and bio-ink.
In the preferred embodiment the holding vessel will include a pin molding system capable of providing structural support that can be manipulated. This molding method will be beneficial in post processing fusion, retraction, remodeling and compaction of printed soft tissue constructs because for a printed tissue construct or organ to be fabricated to the desired mature size and shape it will initially be larger and in many instances have a slightly different shape.
When the construct is matured to a desired state it will be removed from its umbilical and surgically implanted into a recipient.
ALTERNATIVE EMBOBIMENTS
In one alternative embodiment the methods described will be used to create products consisting of biological materials integrated with non-organic materials such as electronic devices and computer components. The methods described in the present invention being a means of storing and/or transporting the integrated organic/electronic materials.
In another alternative embodiment the present invention's ex vivo human perfusion methods could assist with donor organ care. As an example if patient A lives in California and needs a kidney and patient B lives in Boston and needs a kidney, we could have the following scenario. Donor organs become available, but Organ # 1, in California is a poor match for Patient A and Organ #2 in Boston is a poor match for Patient B. Patient A has a family member or friend that is willing to perfuse the kidney while traveling to Boston. Patient B has a family member or friend that is willing to peruse the kidney while traveling to California.
Both patients receive kidneys that may have otherwise gone to waste, been damaged due to ischeniia poor preservation or any other number of reasons.
It does seem like a lot to ask of a friend or family member, but it seems like a more practical scenario than asking a living friend or family member to go into surgery and give up one of their kidneys forever, which is a. relatively frequent procedure.
In another alternative embodiment the present invention will utilize genetically altered animals for assistance with the maturation of tissue constructs or for perfusing tissue selections or organs.
When organs are transplanted between species, immune attack is swift and severe. Pigs for example and other animals have a specific sugar not present in humans and old-world primates. So when a pig organ is transplanted into a baboon, for example, antibodies circulating in the baboon's blood immediately swarm and attack the pig tissue, leading to the death of the organ.
As one example, scientists (particularly David Sachs, the director of the Transplantation Biology Research Center at MGH) made a major advance in overcoming this immune barrier in 2002 by creating genetically engineered pigs that lack the enzyme that attaches the sugar to the surface of, pig cells. In a paper published in Nature Medicine, Sachs showed that baboons given kidneys from these genetically modified pigs lived for up to 83 days, far longer than the average 30-day survival time for animals receiving regular pig kidneys.
The tissue selection is attached to a swine designed to lack an immune system in a surgical process. The tissue selection remains in a system for ex-vivo organ care at near-physiologic conditions, but is also attached to a swine by means of an umbilical cable.
This procedure allows for many beneficial outcomes, such as providing a preferred environment for organ repair, maturation, transport and the use of an animal rather than a human for the perfusion of the tissue selection or organ.
Canada Canada IILIIiIIiIIIIIII~IIIIIIIIIIiIIIIIIIIIII~IIIIIIIILIIIIIilllill II 041- 11 CPO oPiC 19880550 Patent Application of John Archie Gillis for TITLE: METHODS OF PRESERVATION, FABRICATION AND PERFUSION OF
TISSUE CONSTRUCTS
APPLICATION NUMBER 2,685,964 CROSS-REFERENCE TO RELATED APPLICATIONS Not Applicable FEDERALLY SPONSORED RESEARCH Not Applicable SEQUENCE LISTING OR PROGRAM Not Applicable BACKGROUND OF THE INVENTION-FIELD OF INVENTION
The present invention relates to methods of fabrication, preservation, banking, transporting, vascularization and perfusion of tissue constructs.
BACKGROUND OF THE INVENTION-PRIOR ART
Tissue engineering in its early days was considered a sub-field of biomaterials. It has recently grown in both importance and potential and is now considered to be a field of its own.
It generally uses a combination of cells, engineering, materials methods, and suitable biochemical and physio-chemical factors to improve or replace biological functions. Tissue engineering is usually describes as an interdisciplinary field incorporating elements of engineering, material and life sciences.
Most recently tissue engineering has begun to incorporate elements of computer aided design and rapid prototyping. The names currently most in use are bioprinting and organ printing.
Tissues are often fabricated in the laboratory using stem cells, growth and differentiation factors, biomaterials, printing devices and biomimetic environments. It is with these combinations of engineered extracellular matrices (or scaffolds), cells, and, biologically active molecules that researchers in this field have propelled this area of research forward.
One of the major challenges facing tissue engineering today is the requirement for more complex functionality. For a greater number of tissue engineered structures to be considered useful in areas such as transplantation, more biomechanical stability is required along with an advanced means of supplying these structures with nutrients, especially when discussing thick tissue structures.
A cryoprotectant is a substance that is used to protect biological tissue from freezing damage. This damage often occurs due to the formation of ice. Cryoprotectants in common use include glycols, such as ethylene glycol, propylene glycol and glycerol and dimethyl sulfoxide (DMSO), 2-methyl-2, 4-pentanediol (MDP) Sucrose and Trebalose.
Cryobiologists have been using both glycerol and dimethyl sulfoxide for decades to reduce ice formation in sperm and embryos that are cold-preserved in liquid nitrogen.
Mixtures of cryoprotectants have less toxicity and are more effective than single-agent cryoprotectants. A mixture of formaxnide with DMSO, propylene glycol and a colloid, was for many years the most effective of all artificially created cryoprotectants.
Cryoprotectant mixtures have been used for vitrification, i.e. solidification without any crystal ice formation.
Vitrification has important application in preserving embryos, biological tissues and organs for transplant. Vitrification is also used in cryonics in an effort to eliminate freezing damage.
Some cryoprotectants function, by lowering a solution's or a material's glass transition temperature. In this way, the cryprotectants prevent actual freezing, and the solution maintains some flexibility in a glassy phase.
Vitrification techniques utilize low toxicity solutions and optimized cooling and warming curves that, when applied under sterile conditions, allow for better, longer, safer and more convenient storage of complex living systems.
An example of a method of cryopreservation of tissues by vitrification is Khirabadi; Bijan S., Song; Ying C., Brockbank; Kelvin G. M. "Method of cryopreservation of tissues by vitrification", Organ Recovery Systems, Inc. US 7,157,222, (2007) or US
6,740,484 This prior art teaches a method that includes vascularized tissues and avascular tissues, or organs. The method comprises immersing the tissue or organ. in increasing concentrations of cryoprotectant to a cryoprotectant concentration sufficient for vitrification;
rapidly cooling the tissue or organ to a temperature between -80° C. and the glass transition temperature (Tg); and further cooling the tissue or organ from a temperature above the glass transition temperature to a temperature below the glass transition temperature to vitrify the tissue or organ.
This prior art also describes a method for removing a tissue or organ, from vitrification in a cryoprotectant solution. The method comprises slowly warning a vitrified tissue or organ in the cryoprotectant solution to a temperature between -80° C. and the glass transition temperature; rapidly warming the tissue or organ in the cryoprotectant solution to a temperature above -75° C.; and reducing the concentration of the cryoprotectant by immersing the tissue or organ in decreasing concentrations of cryoprotectant.
With this method for treating tissues or organs, viability is retained at a high level. For example, for blood vessels, the invention provides that smooth muscle functions and graft patency rate are maintained.
These and similar methods are great for protecting existing and fabricated tissues from damage, but are not always successful at penetrating deep into thick tissue constructs. These methods have not been used in tissue engineering processes such as those described by the present invention. It is an object of the present invention to prepare cellular compositions with both intracellular and extracellular cryoprotectant solution mixtures prior to a bio printing process, thus allowing precise placement of solutions. In fact cryoprotectants are rarely if ever used in tissue engineering. Most cryoprotectants have been used in protecting existing structures. It can be very difficult to position the protective solutions deep within these already existing structures. This ability of the protective solutions to be selectively located is one of the key benefits of the described invention.
Preservation of organs and tissues are commonplace in medicine, but because organs are most often donated rather that fabricated it can be difficult to place these solutions in areas that can deeply penetrate the structure, especially if the tissue or organ is a thick structure.
Organ printing is usually assisted by computers, dispenser-based, and has an emphasis on three-dimensional fabrication. These methods are aimed at constructing functional organ modules however at present there has been limited success and the printing of entire organs layer-by-layer has not yet been. realized.
Bio-printing or organ printing is a new area of research and engineering that involves printing devices that deposit biological material. Examples of bioprinter technologies would be those in development by Organovo and fabricated at Inventech, which use combinations of "bio-ink" and "bio-paper" to print complex 3D structures.
A number of developments have been occurring in the field of organ printing.
One such development is that of Self-Assembling Cell Aggregates. Forgacs; Gabor;
(Columbia, MO) ;
Jakab; Karoly; (Columbia, MO) ; Neagu; Adrian; (Columbia, MO) ; Mironov;
Vladimir; (Mount Pleasant, SC)"Self-Assembling Cell Aggregates and Methods of Making Engineered Tissue Using the Same", The Curators of the Univeristy of Missouri, Columbia MO, US20080070304,2008 This prior art describes a composition comprising a plurality of cell aggregates for use in the production of engineered organotypic tissue by organ printing. In a method of organ printing, a plurality of cell aggregates are embedded in a polymeric or gel matrix and allowed to fuse to form a desired three-dimensional tissue structure. An intermediate product comprises at least one layer of matrix and a plurality of cell aggregates embedded therein in a predetermined pattern. Modeling methods predict the structural evolution of fusing cell aggregates for combinations of cell type, matrix, and embedding patterns to enable selection of organ printing processes parameters for use in producing an. engineered tissue having a desired three-dimensional structure.
Another development is the method of forming an array of viable cells developed by James Yoo, Tao Xu and Anthony Atala which decribes a method wherein at least two different types of viable mammalian cells are printed on to a substrate. Inventors:
James Yoo, Tao Xu, Anthony Atala. Application number: 12/293,490 Publication number: US
A I Filing date: Apr 20, 2007 These methods of tissue engineering still suffers from some of the limitations of traditional scaffolding methods. There have been some great successes with this method, but the issue of nutrient delivery is still a major, concern.
A common problem with thick tissue structures is that cells deep inside the structure are damaged due to a lack of nutrient delivery. One can delay this problem for a short by preserving the tissue with. a cryoprotectant solution, but unless the tissue is prepared as described in the present invention the problems of getting cryoprotectant solutions into all the desired locations, including cells deep within the structure remains a large and limiting problem.
If tissue engineering is ever to surpass the tissue thickness limit of 100-200 m, it must overcome the challenge of creating functional blood vessels to supply cells with oxygen and nutrients and to remove waste products.
A major dilemma with most current tissue engineering technologies is that most tissues and organs require vascularization and perfusion to survive. Creating this vascular supply and more viable methods of perfusion to a thick-engineered tissue construct remains one of the great challenges in the field today.
SUMMARY
By immersing cells and cellular aggregates, in gradually increasing cryoprotectatnt solutions prior to their dispensing from a three dimensional printing technology we can create a structure that is very well prepared for preservation.
Once cooled the tissues can be transported or banked for drug testing, cell therapies, graphs and implantation, reconstructive surgery, wound healing, cardiovascular treatment and many others beneficial applications.
When the constructs are taken out of their preserved state they will be moved from their vat and into a new holding vessel. The holding vessel will have holes for transporting substances; will contain bioreactor and perfusion bioreactor components, a temperature specific environment and electronic pin molding capabilities.
The tissue selection located in, the holding vessel will then be attached to a circulatory system of a human by connecting the vasculature of the human to an umbilical cord. The other and of the umbilical will be attached to the vasculature of the tissue selection. A tube casing containing a protective solution will protect the cord.
BREIF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a flow chart describing how one or more cells may be prepared for bio-printing and dispensed as a cellular composition that is ready for cryopreservation.
Fig. 2 is a flow chart describing how a number of tissue constructs prepared for preservation can be placed in a vat, preserved, stored and/or transported and warmed.
Fig. 3 shows what could be separate sections of a printed heart valve.
Fig. 4 shows what the separate sections of a printed heart valve could look like if stacked together in a holding vessel.
Fig. 5 shows what the separate sections could look like once fully self assembled into a finished structure.
Fig. 6 shows what a pin molding system capable of adjusting to different shapes for compensating tissue compaction, maturation and movement could look like. The mold would also be helpful with supporting materials involved in extracellwar matrix maturation.
Fig. 7 shows a human being perfusing a. tissue structure or organ by means of attachment to his/her circulatory system. The holding vessel is a Transm.edic device with the organ enclosed inside and attached to the human via an umbilical cable donated from a new born child and enclosed in a protective tube.
DRAWINGS-Reference Numerals - Cryoprotectant Solution 12 - One or more cells 14 - Preparation of cells for preservation 16 Bio-paper 18 - Cryo-prepared cells assembled into self assembling tissue spheroids/bio-ink - Other materials 22 - Dispensing system 24 - Output from dispensing system containing spheroid shaped cryo-prepared cellular compositions situated for the process of self assembly 26 - Tissue Construct #1 28 - Tissue Construct #2 - Tissue Construct #3 32 - Vat 34 - Means of cooling a tissue selection 36 - Means of storage and transport 38 - Means of warming a tissue selection 40 - Means of transferring a tissue selection into a molding system 42 - Section. or layer of a tissue selection to be assembled into a larger structure.
44 - Large tissue structure fabricated from smaller portions 46 - One pin of a pin mold 48 - Pin mold pin holding portion 50 - Protrective holding vessel for tissue selection(s) 52 - Holes for nutrient, blood and supply delivery 54 - Vasculature 56 - Organism with circulatory system that will. supply nutrient delivery and waste removal to a tissue selection.
58 - Transmedic style holding vessel 60 - Protective tube that holds umbilical cord DETAILED DESCRIPTION OF THE DRAWINGS
Fig. 1 is a flow chart showing cryoprotectant solutions 1.0 and one or more cells 12 coming together wherein they are provided with a means of being prepared for preservation 14. The prepared cells of 14 are assembled into self-assembling tissue spheroids or what is known in the art as bio-ink IS. Loaded into a dispensing system 22 are the bio-ink 18, the bio-paper 16 and other materials 20 which may include other cryoprotectant solution, matrix materials, scaffolds and gels. From the dispensing system 22 we get an output containing spheroid shaped cryo-prepared cellular compositions situated for the process of self-assembly 24 into a desired shape, pattern or three dimensional structure.
Fig. 2 is a flow chart showing a number of different tissues 26, 27, 28, which will be loaded into a vat 32 with a shape complementary to the shape of the printed tissue selections.
A means of cooling 34 will be provided and when cooled to a desired temperature the tissues will be stored and/or transported 36. When the tissues reach their location or it is desired to remove them from their cryopreserved state a means of warming 3 8 will be provided so as to enable transfer to a pin molding system 40 or for other uses.
Fig. 3 is a diagram of a heart valve printed in layers or sections 42. Each section 42 was printed as a separate unit with a specific shape. At this stage of the process we can see how when the layers are placed together that they will form. the shape of a heart valve.
Fig. 4 is a diagram of our layers 42 stacked together to form a structure 44 that will be coaxed into self assembly and form the shape of a heart valve.
Fig. 5 shows what separate sections of a heart valve could look like once fully assembled into a finished structure 46.
Fig. 6 provides a visual example of what a pin molding system capable of adjusting to different shapes for compensating tissue compaction, maturation and movement could look like. 42 shows a holding vessel that has a means to provide a protective environment for the tissue selections. In the preferred embodiment it will be a membrane like encasing made from a material that is flexible, malleable and capable of a variety of shapes so that when self assembly, fusion, maturation, compaction or change in shape occurs to the construct our pins 46 can move to compensate for these changes and in most instances prevent unwanted areas of the structure from moving into undesirable locations. 48 is a stand for holding the pins of our mold. 46 shows pins that when moved together can create a desired shape or mold that can change over time for allowing the same casing or holding vessel the ability to provide structural support during changes to the structure. Holes 52 are provided to the holing vessel to allow transport of materials to and from the tissue selection(s) from an outside source.
Vasculature 54 is shown passing through the holes. The vertical three-dimensional image screen described in patent number: 4654989 Filing date: Aug 16, 1.985 Issue date: Apr 7, 1987 descibes a screen with ability to mold to many objects. United States Patent 6,625,088 to Mah; Pat Y. and Tinier; Robert Bruce issued on September 23, 2003 describes a similar pin display device that has an electronic mechanisms for moving its pins into different shapes.
Fig. 7 shows a human being or patient 56 perfusing a tissue structure or organ by means of attachment to their circulatory system. The holding vessel is a Transmedic device 58 with the organ enclosed inside and attached to the human via an u#iobilical cable donated from a new born child and enclosed in a protective tube 60.
DETAILED DESCRIPTION OF PREFERRED EMBODNTS
Reference will now be made in detail to various embodiments of the invention, one or more examples of which are set forth below. Each embodiment is provided by way of explanation of the invention, not limitation, of the invention. In, fact, it will be apparent to those skilled in the art that various modifications and variations may be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, may b used in another embodiment to yield a still further embodiment. Thus, it is intended that the present invention cover such modifications and variations as come within the scope of line appended claims and their equivalents.
In the preferred embodiments the present invention describes a number of steps for the fabrication, preservation and perfusion of a tissue constru t.
The process begins by immersing cells in varying level) s of cryoprotectant solutions. The cells are then aggregated into self-assembling tissue spheroids, and dispensed into a desired shape.
The innovative method comprises ink jet printing a cei composition onto a substrate wherein the cells within the composition have been prepar~d for cryopreservation, cooling, freezing or vitrification. A great example of Ink jet printing of viable cells is US Patent 7,051,654 Boland; Thomas (Suwanee, GA), Wilson, Jr.; illiam Crisp (Easley, SC), Xu; Tao (Clemson, SC), which, is hereby incorporated by reference in its entirety. It describes a method for forming an array of viable cells. In one embodi' cnt, the method comprises ink jet printing a cellular composition containing cells onto a substrate. Upon printing, at least about 25% of the cells remain viable after incubation for 24 hour at 37° C.
in a 5%
CO2/95% O2 environment.
In the preferred embodiment the cultured cells that are included in the cellular composition to be printed are prepared with. varying levels of cryoprotectant solutions. A
variety of solutions can be used to generate various levels of results and successes.
Examples of some potential methods that may be used in whole or in part include, but are not limited to "Method of cryopreservation of tissues by vitrification" (Khirabadi; Bijan S., Song;
Ying C., Brockbank; Kelvin G. M. "Method of cryopreservation of tissues by vitrification.", Organ Recovery Systems, Inc. US 7,157,222, 2007), The cryogenically prepared cells will form a. bio ink that will be loaded into a three dimensional fabrication device. A great example of a bio ink is US Patent Application 20080070304 to Forgacs; Gabor; (Columbia, MO) ; Jakab; Karoly; (Columbia, MO) ; Neagu;
Adrian; (Columbia, MO) ; Mironov; Vladimir; (Mount Pleasant, SC) "Self Assembling Cell, Aggregates and. Methods of Making Engineered Tissue Using the Same", which is hereby incorporated by reference in its entirety and explains bio ink and bio paper.
No prior art reference provides a description of a process incorporating the use of cryogenic preparation of cells or cell aggregates for the purpose of being loaded into a printer.
This is one of the novel features of the present invention. With prior methods of applying cryoprotectant solutions to some tissue constructs, (especially into think constructs or organs) it has been found difficult if not impossible to get the cryoprotectant solutions to the desired locations. The present invention provides a remedy for this problem.
After being dispensed from an ink jet printer the cellular spheroids or aggregates will be preserved by methods of freezing or vitrification. The construct will be stored and transported for cell therapies, drug tests, and in the preferred embodiment oftbe present invention used, as a section to be fused with other similar sections to create a larger construct.
The prepared cells are then placed into a vat or similar cooling device, next to one or more other cellular constructs that have been prepared in a similar manner. The constructs are then.
vitrified or frozen. The cells may now be banked or transported for drug testing or cell therapies and once taken out of their preserved state they will be coaxed into self=assembly and fused together to create a larger structure.
One of the reasons the present inventor feels it is pertinent to create and preserve the tissues in sections is so that they can be easily stored at a later time when they are needed, and so that the bioprinting system is freed up for use. Another reason for printing the constructs in.
sections is that some bioprinters will be designed and/or set up for the creation of specific tissue structures. As an example; bioprinter I would be programmed to print section A of a kidney with specific materials and preservation solutions and thus the technician is trained to know exactly what is required each time, while bioprinter 2 is programmed to print section B
of the same kidney with their different but specific materials and preservation solutions. The process continues with bioprinter 3 doing section C and so on., until all the required parts are constructed. By printing the sections simultaneously we can decrease the time it takes to complete the more elaborate structure.
In the preferred embodiment the printers will be programmed to complete the sections at the same time so that they can be placed into a vessel for preservation. Once they are preserved they can be transported to the geographic location in which they are needed.
A viable method of transportation is also a. very beneficial, outcome of the present invention. Organs and tissue assays do not last long if not protected properly.
Bioprinting labs are very expensive and require expertise in operation. As an.
example Inventec sells their bioprinters for $250,000.00 a pretty steep price for any lab. These labs and expertise are only located in a few geographic locations, but with the described preservation methods, preserved tissues fabricated with computer and roboticially targeted.
precision will make it much more viable for the transportation of these products for their use in cell therapy products, tissues, organs and tissue engineered constructs.
The cryogenically prepared cells are printed in layers, and as the layers are completed they are mechanically lowered into a vat, and put into a vitrified or frozen state.
The layers are organized so that they fit together in a desired shape or pattern that will allow the proper portions to fuse in the correct areas when taken out of their vitrified or frozen states. An example of this is to position a vascular network such that when cellular adhesion (self assembly) occurs it will become one unit and thus when taken out of a vitrified or frozen state the sections will fuse together in the correct and desired locations, and be ready to be placed. in a device capable of providing the required supply of nutrients and materials.
Vasculature has been bioprinted in the labs of Anthony Atala without cryopresevation included to a limited degree. It has also been successfully attached to a perfused bioreactor.
In a preferred embodiment self-assembly may occur after preservation, however in alternative embodiments it will occur prior to preservation.
When the individual sections are placed into a vat, the vat will be shaped as to support the dimensions of the larger structure to be fabricated or preserved. At this point the structures will have been persevered or will undergo a preservation process. The construct is now transported to its required location.
When the structure reaches its destination it is taken out of its state of preservation. At this point the structure can be carefully removed from its vat into a holding vessel with electronically programmable and movable pin molding capabilities for supporting the structures and for also providing support for a protective membrane that will encase the structure in a protective biological environment. This type of protective pin molding biological environment will provide support for the structure yet allow for changes to occur during post processing fusion, retraction, remodeling and compaction. Another object of the holding vessel is its temperature specific environment.
When the structures are taken out of their vitrified state they will be coaxed into self assembly as is described in US Patent Application 20080070304. The self assembling tissue spheroids of each section may have aggregated into a larger tissue structure prior to preservation and thus these fused tissue sections will aggregate to form yet an even larger structure. In another embodiment the smaller sections will also require time for self assembly.
There are a number of instances where the present methods of preservation will be very useful. if the tissue constructs are not needed for some time after printing or if they are required to be transported the present methods will assist with preventing damage and cell death from occurring. Often tissues are transported long distances for drug testing, cell therapies and if there are no bio-printing laboratories near an area where bioprinted structures are wanted or needed, the aggregation methods explained herein may be a very necessary requirement.
Another reason that the described method is practical is the high cost and low'success rate of many other alternatives. Time is of the essence when printing tissue constructs, as they can only be maintained for relatively short periods of time after, printing, before damage occurs.
The present sectioning method can be used without the use of cryogenic solutions integrated into the construction process, but only if a company has a number of bioprinters printing sections of a predetermined structure at the same time. This type of lab would then need to stack the sections into a holding vessel. and perfuse them with nutrients as soon as possible. This method is not practical if the construct is to be transported.
The structures will be provided with nutrients and waste removal using standard methods found in the art, such as bio reactors, perfused bioreactors or solutions used in systems for ex vivo care at new physiologic conditions, however once a vascular structure or vascular system, such as a bioprinted intraorgan branched vascular system has been assembled and becomes mature and functional enough for initiation of intravascular perfusion it will be attached to an umbilical cable. The umbilical cable may be fabricated from human cells or may be one donated by a suitably matched new born baby and then attached to a human circulatory system. This would likely be the circulatory system of the future recipient of the structure.
A group from South Carolina as well as a group led by Gabor Forgacs' have recently demonstrated that building a branching intraorgan vascular tree is a realistic and achievable goal. This issue was also addressed by Peter Wu (University of Oregon, USA) who presented applications of LAB in fabricating branch/stem, structures with human endothelial cells and T
Boland who presented results on thermal inkjet printing ofbiomaterials and cells for capillary constructs. (Cui X and Boland T 2009 Human microvasculature fabrication using thermal ink jet printing technology Biomateri.als 30 6221-7) When the structure has fused into a single unit it will remain in its holding vessel where it will continue to receive nutrients and blood from the human circulatory system. This system.
will also provide the structure with the ability to remove waste.
One of the great benefits of the structure being located outside of the body is that it may be tended to by doctors, engineers and other professionals for other additional procedures, tests or substance delivery that may be beneficial to the survival and maintenance of the structure.
The structure may when required also receive external, electrical stimuli.
Other great benefits of the structure being perfused by the patient's own circulatory system, yet essentially being located outside the body is that it can be accessed, repaired, manipulated and supplied with additional substances or therapies.
Current methods of perfusing a tissue structure are limited, due to time constraints. This is seen in cases of organ donation. When a donated organ is matched with a recipient, it is imperative that the organ reaches the recipient in as short of time as possible. Even with our advanced technologies, helicopters and database matching systems organs are often lost, due to a variety of reasons that include injuries during brain-death, ischemia, cell death and other causes.
Currently there are a number of systems that are perfusing organs such as Transm.edics, "Organ Care System", Organ Recovery Systems "LifePort" technologies and the Toronto XVIVO Lung Perfusion System. The Lung Perfusion Systern is being worked on by Dr. Shaf Keshavjee in the Lung Transplant Program at Toronto General Hospital (TGIF).
They have developed an "ex vivo" or outside the body technique capable of continuously perfusing or pumping a bloodless solution containing oxygen, proteins and nutrients into injured donor lungs. This technique allows the surgeons the opportunity to assess and treat injured donor lungs, while they are outside the body, to make them suitable for transplantation.
These methods of perfusion are great advances in medical technologies, but still have their limitations. The present invention describes that at first seems odd, but is actually the most natural method of perfusing either a transplanted organ or a tissue engineered construct. If we think of how a fetus is perfused in the womb we have a fetus attached to an umbilical cord, which, is attached to its mother. Both the fetus and the umbilical are in a protective solution.
In the present invention we create something very similar. Our fetus is out tissue engineered construct and our mother is the person who will be having the construct or organ implanted into them. In the preferred embodiment the ex vivo perfusion module will be attached via existing or fabricated umbilical cables to the construct or organ to be perfused. The construct or organ will be located outside of the body and housed in a protective temperature specific environment, likely at 37 degrees C and may include a protective solution for, surrounding the construct/organ. The tube attaching to the recipients circulatory system via an umbilical cable will be housed in a tube containing a protective solution, which may contain Wharton's Jelly or a suitable substitute, nutrient composition, or liquid that may assist in sustaining the cord during perfusion of the construct. Connection of this cord will require surgical attachment.
In placental mammals, the umbilical cord (also called the birth cord or funiculus uznbilicalis) is the connecting cord from the developing embryo or fetus to the placenta.
During prenatal development, the umbilical cord comes from the same zygote as the fetus and (in humans) normally contains two arteries (the umbilical arteries) and one vein (the umbilical vein), buried within Wharton's jelly. The umbilical vein supplies the fetus with oxygenated, nutrient-rich blood from the placenta. Conversely, the umbilical arteries return the deoxygenated, nutrient-depleted blood.
Successful perfusion of an extra organ using a similar procedure in vivo has been accomplished in the art by what is known as hetcrotopic surgery. In this medical procedure the patient's own heart is not removed before implanting a donor heart. The donor heart is 1.7 positioned so that the chambers and blood vessels of both hearts can be connected to form what is effectively a'double heart'.
Another example of in vivo perfusion of an extra organ is that of a kidney transplant. In many kidney transplants the original but likely damaged kidneys are left in the recipient.
An example of ex vivo perfusion is that of babies who are occasionally born with organs outside their body and often survive this way for many months prior to having the organs transferred inside their body.
Where the present invention differs from these procedures is that the heterotropic procedure takes place inside an. organism not via an, ex vivo attachment.
Another difference is that in the present invention the organism that the construct or organ is first attached to after being printed acts as a temporary lobby area or location and once the organ has been matured it will be attached to the recipient The tissue constructs of the present invention include portions of, or whole tissues (i.e., bone, cartilage, blood vessels, bladder, etc.) The tissue harvested may consist of any biological material and may include materials that have been manipulated and/or changed from their original state, such as geneticially altered materials or stem cell cultivations.
The dispensing systems of the present invention include computer aided design, manufacturing, assembly and/or printing systems. These systems make use of computer technology to aid in the design, manufacturir, assembly and/or printing of a product.
Examples of such systems include, Direct Digital Manufacturing, Rapid Prototyping, Three Dimensional Printing, Bio-printing, (CAD/CAM), Stereolithography, Solid Freeform Fabrication, Self-Replicating Machines, 3D Microfabrication, Digital Fabrication and Desktop Manufacturing Systems, and the methods and technologies involved, developed and understood by those skilled in the art.
The Bio-printing systems of the present invention will include the use of what is known in the art as bio-paper and bio-ink.
In the preferred embodiment the holding vessel will include a pin molding system capable of providing structural support that can be manipulated. This molding method will be beneficial in post processing fusion, retraction, remodeling and compaction of printed soft tissue constructs because for a printed tissue construct or organ to be fabricated to the desired mature size and shape it will initially be larger and in many instances have a slightly different shape.
When the construct is matured to a desired state it will be removed from its umbilical and surgically implanted into a recipient.
ALTERNATIVE EMBOBIMENTS
In one alternative embodiment the methods described will be used to create products consisting of biological materials integrated with non-organic materials such as electronic devices and computer components. The methods described in the present invention being a means of storing and/or transporting the integrated organic/electronic materials.
In another alternative embodiment the present invention's ex vivo human perfusion methods could assist with donor organ care. As an example if patient A lives in California and needs a kidney and patient B lives in Boston and needs a kidney, we could have the following scenario. Donor organs become available, but Organ # 1, in California is a poor match for Patient A and Organ #2 in Boston is a poor match for Patient B. Patient A has a family member or friend that is willing to perfuse the kidney while traveling to Boston. Patient B has a family member or friend that is willing to peruse the kidney while traveling to California.
Both patients receive kidneys that may have otherwise gone to waste, been damaged due to ischeniia poor preservation or any other number of reasons.
It does seem like a lot to ask of a friend or family member, but it seems like a more practical scenario than asking a living friend or family member to go into surgery and give up one of their kidneys forever, which is a. relatively frequent procedure.
In another alternative embodiment the present invention will utilize genetically altered animals for assistance with the maturation of tissue constructs or for perfusing tissue selections or organs.
When organs are transplanted between species, immune attack is swift and severe. Pigs for example and other animals have a specific sugar not present in humans and old-world primates. So when a pig organ is transplanted into a baboon, for example, antibodies circulating in the baboon's blood immediately swarm and attack the pig tissue, leading to the death of the organ.
As one example, scientists (particularly David Sachs, the director of the Transplantation Biology Research Center at MGH) made a major advance in overcoming this immune barrier in 2002 by creating genetically engineered pigs that lack the enzyme that attaches the sugar to the surface of, pig cells. In a paper published in Nature Medicine, Sachs showed that baboons given kidneys from these genetically modified pigs lived for up to 83 days, far longer than the average 30-day survival time for animals receiving regular pig kidneys.
The tissue selection is attached to a swine designed to lack an immune system in a surgical process. The tissue selection remains in a system for ex-vivo organ care at near-physiologic conditions, but is also attached to a swine by means of an umbilical cable.
This procedure allows for many beneficial outcomes, such as providing a preferred environment for organ repair, maturation, transport and the use of an animal rather than a human for the perfusion of the tissue selection or organ.
Claims (30)
1. A method of producing a tissue construct prepared for preservation at low temperatures comprising, the dispensing of a cellular composition containing at least one cell with at least one cryoprotectant solution from a dispensing system and providing a means for self assembly for one or more cellular compositions to fuse into a larger tissue construct.
2. The method of claim 1 wherein said cellular composition containing at least one cell with said at least one cryoprotectant solution is prepared for cryopreservation prior to dispensing.
3. The method of claim 1 wherein said cellular composition containing at least one cell with said at least one cryoprotectant solution is prepared for cryopreservation after being released from said dispensing system.
4. The method of claim 1 wherein said dispensing system comprises a selection of computer aided design, manufacturing and assembly systems, ink-Jet printers, bio-printing and organ-printing systems.
5. The method of claim 1 wherein said cellular composition consists of one or more self-assembling tissue spheroids.
6. The method of claim 1 further including said one or more cells being prepared with varying levels of cryoprotectant solutions before placing them into said dispensing system.
7. The method of claim 1 wherein said cryoprotectant solution is any substance that is used to protect biological tissue from freezing damage.
8. The method of claim 1 wherein said dispensing system further includes one or more separate cartridges for different cryogenically prepared cells, cryoprotectant solutions, growth factors, matrix materials, nutrients, hydrogen sulfide, lithium and other supplies.
9. A method of preserving, transporting and joining a plurality of tissue constructs prepared for preservation at low temperatures with cryoprotectant solutions comprising:
(a) placement of said tissue constructs into a holding vessel suitable for temperature specific environments, (b) a means of cooling said tissue constructs for a period of time, (c) a means of storage and transportation for said tissue constructs, (d) warming said tissue constructs and (e) providing substance delivery and an environment suitable for the adhesion of said tissue constructs.
(a) placement of said tissue constructs into a holding vessel suitable for temperature specific environments, (b) a means of cooling said tissue constructs for a period of time, (c) a means of storage and transportation for said tissue constructs, (d) warming said tissue constructs and (e) providing substance delivery and an environment suitable for the adhesion of said tissue constructs.
10. The method of claim 9 further including means for cooling said tissue constructs that includes methods of solidifying, freezing and vitrifying said tissue constructs.
11. The method of claim 9 wherein said holding vessel is a mold of a specific shape and capable of providing structural support when tissue sections are warmed.
12. The method of claim 9 wherein said holding vessel is a vat.
13. The method of claim 9 wherein said holding vessel is a vat which incorporates a mold of a specific shape and is capable of providing structural support when tissue sections are warmed.
14. The method of claim 9 wherein said holding vessel contains a substance capable of cooling said one or more tissue constructs after dispensing.
15. The method of claim 9 wherein said. holding vessel may further comprise one or more holes for releasing materials such as cryoprotectant solutions, matrix gels, and any unwanted support materials and for receiving materials such as, nutrients, blood, blood vessels, an umbilical cable and substances which promote angiogenesis, which include VEGF and other known growth factors (TGF, PDGF, VGF).
16. The method of claim 9 wherein said holding vessel comprises bioreactor components and methodologies that include a means for a variety of temperature specific environments.
17. The method of claim 9 wherein the device providing substance delivery and an environment suitable for the adhesion of said tissue constructs comprises a perfusion device.
18. A method of providing substance transfer for at least one tissue selection comprising;
(a) a means of attaching said tissue selection to the circulatory system of a living organism ex vivo (b) a means of attaching at least one cord with means capable of delivering substances to said tissue selection from said circulatory system and (c) a means of attaching at least one cord with a means capable of removing unwanted substances from said tissue construct and (d) a protective holding vessel for said tissue selection
(a) a means of attaching said tissue selection to the circulatory system of a living organism ex vivo (b) a means of attaching at least one cord with means capable of delivering substances to said tissue selection from said circulatory system and (c) a means of attaching at least one cord with a means capable of removing unwanted substances from said tissue construct and (d) a protective holding vessel for said tissue selection
19. The method of claim 18 wherin said means of attaching said tissue selection to said circulatory system of a living organism is accomplished by the use of an umbilical cord with means capable of transporting nutrients, blood supplies, growth factors, amino acids, electrolytes, gases, hormones, blood cells and other organic materials.
20. The method of claim 18 wherein said cord is an umbilical cable selected from a living organism, engineered with tissue engineering methods, or a non organic unit.
21. The method of claim 20 wherein said cord is immersed in a protective solution and tube.
22. The method of claim 21 wherein said protective tube contains a selection of Wharton's Jelly, nutrients, and other protective substances.
23. The protective holding vessel of claim 18 further comprising a means for substance delivery, substance removal and a temperature specific environment
24. The protective holding vessel of claim 23 wherin said holding vessel is selected from a group of devices in whole or in part which are used in the perfusion of tissues and organs.
25. The method of claim 18 wherein said tissue selection is a donor organ.
26. The method of claim 18 wherein the living organism is a human being
27. The method of claim 18 wherein the living organism is an animal
28. The method of claim 27 wherein the animal is genetically modified.
29. The method of claim 28 wherein the animal is genetically modified to lack an immune system.
30. A method of supporting a tissue engineered structure comprising the use of a pin molding system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2685964 CA2685964A1 (en) | 2009-11-12 | 2009-11-12 | Methods of fabrication and vascularization of thick three dimensional tissue constructs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2685964 CA2685964A1 (en) | 2009-11-12 | 2009-11-12 | Methods of fabrication and vascularization of thick three dimensional tissue constructs |
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| CA2685964A1 true CA2685964A1 (en) | 2011-05-12 |
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| Application Number | Title | Priority Date | Filing Date |
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| CA 2685964 Abandoned CA2685964A1 (en) | 2009-11-12 | 2009-11-12 | Methods of fabrication and vascularization of thick three dimensional tissue constructs |
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| CN106163581A (en) * | 2013-11-05 | 2016-11-23 | 哈佛学院院长及董事 | The method printing the tissue construct of the vascular system with embedding |
| US10782217B2 (en) | 2016-07-12 | 2020-09-22 | Deka Products Limited Partnership | System and method for applying force to a device |
| US10808218B2 (en) | 2015-10-09 | 2020-10-20 | Deka Products Limited Partnership | Fluid pumping and bioreactor system |
| US11254901B2 (en) | 2016-07-12 | 2022-02-22 | Deka Products Limited Partnership | System and method for printing tissue |
| US11299705B2 (en) | 2016-11-07 | 2022-04-12 | Deka Products Limited Partnership | System and method for creating tissue |
| US11530380B2 (en) | 2017-07-12 | 2022-12-20 | Deka Products Limited Partnership | System and method for transferring tissue |
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2009
- 2009-11-12 CA CA 2685964 patent/CA2685964A1/en not_active Abandoned
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106163581A (en) * | 2013-11-05 | 2016-11-23 | 哈佛学院院长及董事 | The method printing the tissue construct of the vascular system with embedding |
| CN106163581B (en) * | 2013-11-05 | 2019-10-25 | 哈佛学院院长及董事 | Print the method with the tissue construct of vascular system of embedding |
| US10808218B2 (en) | 2015-10-09 | 2020-10-20 | Deka Products Limited Partnership | Fluid pumping and bioreactor system |
| US10782217B2 (en) | 2016-07-12 | 2020-09-22 | Deka Products Limited Partnership | System and method for applying force to a device |
| US11254901B2 (en) | 2016-07-12 | 2022-02-22 | Deka Products Limited Partnership | System and method for printing tissue |
| US11543336B2 (en) | 2016-07-12 | 2023-01-03 | Deka Products Limited Partnership | System and method for applying force to a device |
| US11299705B2 (en) | 2016-11-07 | 2022-04-12 | Deka Products Limited Partnership | System and method for creating tissue |
| US11939566B2 (en) | 2016-11-07 | 2024-03-26 | Deka Products Limited Partnership | System and method for creating tissue |
| US11530380B2 (en) | 2017-07-12 | 2022-12-20 | Deka Products Limited Partnership | System and method for transferring tissue |
| US11939564B2 (en) | 2017-07-12 | 2024-03-26 | Deka Products Limited Partnership | System and method for transferring tissue |
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