CN1432645A - Human cell bank and its application in autogenous cell transplantation and tissue repair - Google Patents
Human cell bank and its application in autogenous cell transplantation and tissue repair Download PDFInfo
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Abstract
The present invention relates to human cell bank and its preparation process and application, especially a method of collecting, separating, freeze drying and preserving cell and/or tissue of full-tern newborn baby and infant in the hearty life activity stage, preparing the cell and/or tissue, and establishing corresponding cell bank. Furthermore, the present invention relates to the application of the cell bank in autogenous repair, replacement and regeneration in future life course, especially the damage, ageing or hypofunction in his senile stage caused by various diseases, various physical, chemical and biological damage and retrogressive pathological changes.
Description
The present invention relates to people's cell bank, its preparation method and application.More particularly, the present invention relates to, prepare these cells and/or tissue and set up the method for corresponding cell bank at people's FTNB to baby children vital movement is collected, separated vigorous period and freeze-drying is preserved cell and/or tissue.The invention further relates to said cell bank from the body reparation, substitute or regenerate same individuality in following vital process particularly senectitude because of various diseases, various physics or chemistry or biology damage, and the application in damaged, the aging or hypofunction that degeneration caused.
Cell is the fundamental unit of body vital movement.In people's whole vital movement, body is some cell aging and death constantly always, many new cell proliferations and regeneration is arranged to compensate old and feeble and dead cell simultaneously again.Most cells usually can be repaired and be upgraded when being subjected to consuming voluntarily in the body.For a particular individual, even the old and feeble or dead life-span that influences body hardly of local many cells (except that myocardial cell and brain cell).Yet the aging that cell is overall and the aging of whole machine body are again closely-related.So investigators usually start with from cell aging mechanism and carry out geratologous research.
The damage of body pair cell and tissue has huge repairing and restorability, not only can recover the structure of cell and tissue, but also can recover its function in varying degrees.These repair processes are that the division and the propagation of damaged tissue cell are not finished by the part.The extensive clinically at present method of tissue repair that adopts comprises autograft, allosome tissue's transplanting and uses the tissue substituent of natural or chemosynthesis.Though wherein immunological rejection has been avoided in autograft, preceding topic is the healthy tissues that needs the damage human body self.There is histocompatibility for want of in heteroplastic transplantation and the immunological rejection that causes, and the limited problem of donor source.Use artificial tissue substituent equally also to have the foreign matter exclusive problem, and the danger of the infection of causing is arranged.
Foundation and development along with regenerative medicine, possible application cell engineering of scientists and tissue engineering technique, based on matrix rack, corresponding histocyte and three fundamentals of somatomedin, the natural condition of analog machine body tissue organ and developmental biology rule, mix in viable cell, big right cellular constituent, cell synthetic product and the cell and iuntercellular is regulated material etc., with artificial constructed different biological tissue and organ, for example cartilage, bone, tendon, muscle, skin, blood, heart valve and blood vessel etc.Regenerative medicine comprises the regeneration of histoorgan form and two aspects of functional regeneration.In general, people always expect that the regenerated tissue not only can or substitute local cells or the tissue that sustains damage from the morphology reparation, more wishes to recover and to compensate the physiological function of damaged cell or tissue simultaneously.
In recent years, along with Protocols in Molecular Biology and clinical medical continuous progress, this therapy of gene therapy has caused increasing medical research and clinical position person's concern and has played an active part in.At present, occurred many genetic stocks transfer or modification technique being combined with cell engineering or tissue engineering technique, be used to repair or compensate the hereditary defect of local organization or organ or the research report of afunction (backing off) (as referring to United States Patent (USP) 6,077,981,6,103,230,6,264,941 etc.).Yet these technology are very complicated, and required cost is very high, still be in the early stage laboratory study stage at present, and success ratio is very low.
In order to repair and recover the normal morphology and the function of local damaged cell effectively, there be to be solved the use that the favorable tissue consistency is promptly arranged except remaining at present with selection in a large amount of technical problems, again admirably outside the extracellular matrix of simulated in vivo environment (ECM), successfully obtain and cultivate and transplant the seed cell that the back continues to keep its intrinsic form and function, be still problem demanding prompt solution in cell engineering and the field of tissue engineering technology.
An object of the present invention is to provide an autogenous cell storehouse that comprises the cell of preserving by different tissues or organ origin separation, purifying and freeze-drying.
According to a preferred embodiment of the invention, wherein the seed cell of cell bank comprises epidermis epithelial cells, epithelial cell, inoblast, mesenchymal cell, breaks up complete or undifferentiated peripheral blood cells, marrow hemopoietic stem cells, periosteum cell, chondrocyte, cord blood stem cell, chondrocyte, myocyte, immune system cell, epilemma cell, syneytiotrophoblast and hair cell vascular endothelial cell.
According to the preferred embodiments of the invention, the seed cell in the wherein said cell bank derives from newborn infant or its appurtenant, and the infant.
According to the preferred embodiments of the invention, wherein said newborn infant's appurtenant comprises umbilical cord, fetal membrane and placenta.
According to a preferred embodiment of the invention, the seed cell in the wherein said cell bank is an autogenous cell.
According to a preferred embodiment of the invention, the seed cell in the wherein said cell bank through or do not pass through genetic modification.
According to the preferred embodiments of the invention, the seed cell in the wherein said cell bank obtains by the not damaged method basically.
According to a preferred embodiment of the invention, wherein said cell bank was preserved 30 years at least.
Another object of the present invention provides respective organization cell in the cell bank that is defined as above and carry out application in Transplanted cells or the tissue repair after same individuality is grown up or be old.
According to a preferred embodiment of the invention, wherein said application also comprises the host cell that is used for as the gene therapy foreign DNA.
According to the preferred embodiments of the invention, wherein said application also comprises and is used for cosmetic surgery.
Fig. 1 shows the typical paving stone shape cell monolayer (* 100) that the cultivation of the former representative cornu cutaneum of Wistar neonate rat protein cells in vitro grew up to after 10 days.
Fig. 2 shows the intensive cell monolayer (* 100) that Wistar neonate rat corium inoblast vitro culture of former generation grew up to after 7 days.
Fig. 3 shows the scanning electron microscope ultrastructure photo (* 300) of the matrix rack of the multiple layer artificial skin that 2: 1 (v/v) mixtures of use collagen protein-chitin prepare.
Fig. 4 shows that the senile rat skin wound implants the 6th day the histological observation result (HE dyes, * 200) behind the multiple layer artificial skin of body surface skin and dermal cell who collects when be born based on animal.
Fig. 5 shows that the senile rat skin wound implants the 15th day the histological observation result (HE dyes, * 400) behind the multiple layer artificial skin of body surface skin and dermal cell who collects when be born based on animal.
The present invention relates to human cell bank, particularly relate to and comprise and derive from mature new that Fast Growth grows The cell bank of living youngster or its adjunct or infant's linked groups or the cell of organ. The present invention advances one Step relates to the method for preparation said cell and cell bank, and when corresponding individuality after adult or old age, The application of said cell bank cell in autogenous cell transplantation, tissue damage reparation and gene therapy.
In cell and field of tissue engineering technology, present existing key issue comprises the group of seed cell Knitting compatibility, cell behavior feature (comprises synthetic and the extracellular matrix secretion composition, produces justacrine The ability of respective fine intracellular cytokine, cell receptor, iuntercellular medium, signal protein and hormone is in interior each Plant cell function), and cell division and multiplication capacity etc. Although autogenous cell transplantation can be effectively Overcome immunological rejection and relevant adverse immune response problem, but for senectitude particularly of adult later stage Individuality, when needs carry out cell tissue transplant or rebuild new when treating transplanted tissue, one As can only utilize diseases or tissue damage cell and/or tissue at that time. Yet, this moment human body Some is organized the retrogression that takes place in various degree or wears out, and shows as cell viability and multiplication capacity Reduce, and the decline of normal physiological function. For example, the human embryonic fibroblast can go down to posterity 40～60 In generation, birth can cultivate for 20～40 generations by 15 years old, and can only be in 10～30 generations of external biography more than 15 years old. Show that on evidence colony's passage number of embryo fibroblast and the life-span of these species are roughly proportional Relation. In the biological tissue transplantation experiments, also obtained similar result.
The most significant structural change of cell ageing is the minimizing of the interior plasm of cell and moisture content, and cell The minimizing of karyopycnosis and the outer coloring matter of nuclear. Another notable feature of cell ageing is endochrome Or ceroidlike material deposition. For example, the deposition of these materials can show as " senile plaque expelling " in the Skin Cell Formation.
Except said structure and form change, along with cell ageing, in a series of biochemical generations, also take place in cell Thank to the change of ability, particularly show as the amino acid and protein synthesis rate and descend. In addition, aging The activity of desmoenzyme and content also change to some extent, and for example old nerve cell thiamine pyrophosphatase is lived The property reduction causes the transportation function of the secreting function of golgiosome and vesica low. Ling Wai Yi Certificate is real, The telomere length of hematopoietic cell shortens with advancing age, and the telomerase activation raising, thereby Play the effect (Engelhardt M et al, Blood, 90:182-193,1997) that prolongs cell survival.
Only with regard to the skin histology that is easy to most observe and study, it is old skin to occur with age Form and the metabolic characteristics main manifestations changed are that pachyderma, fibroblast lose vigor, collagen closes Become minimizing, elastin laminin content, aminoglycan content to reduce and skin heart minimizing and expansion Deng. Wherein, reduce owing to process relevant enzyme after collagen synthesis and the translation, and cause skin Middle collagen content descends. Because being connected between the minimizing of aging skin medium vessels and epidermis and corium Thickening is so the elderly's skin is easier to suffer mechanical damage. In addition, because can in the elderly's skin Produce and discharge inflammatory cell and the mast cell of lymphokine, interleukins and Porcine HGF Lack, so skin is easier to suffer the infringement of extraneous virulence factor.
On the contrary, the young cell cells that particularly derives from children's foetal period to infancy does not exist State the many disadvantages of aged cells. These cellular metabolisms are vigorous, multiplication capacity is extremely strong, and function is complete Kind. Particularly along with the continuous progress of cytology and cell engineering, not only can separate at present, Purifying obtains and the vigorous young cell of these vitality of long preservation (for example preserving more than 10 years), And external these cells of a large amount of propagation of having the ability fully, and utilize at present ripe with advance one future The technology of step development as a very valuable optimum cell source, is used for structure with these cells Build various artificial organs even organ, the various organs of body and the group that cause to treat various pathogenic factors Damaged, the functional disturbance of knitting and disease.
According to the present invention, the seed cell that is used for setting up cell bank include but are not limited to epithelial cell, Dermal cell, fibroblast, mesenchymal cell, break up complete or undifferentiated PBC, Marrow hemopoietic stem cells, Cord blood " are done " cell, immune system cell, liver cell, epilemma Cell, periosteum cell, cartilage cell, myocyte, syneytiotrophoblast and hair cell blood vessel endothelium are thin Born of the same parents.
According to the preferred embodiments of the invention, wherein said epithelial cell comprises the epiderm skin cell And oral cavity and gastrointestinal tract epithelial cell.
According to the preferred embodiments of the invention, wherein said immune system cell comprises thymocyte And splenocyte.
According to the present invention, comprise FTNB or baby for the cell derived of setting up cell bank of the present invention Child's normal body histocyte.
The term that uses in this specification " neonate " refers to reach 37 thoughtful 42 weeks, body weight pregnant age FTNB more than or equal to the 2500g birth. Children from birth to 1 monthly age are called the neonate Phase. The term " infant " that uses in this explanation refers to the children that were born 1 month to 3 years old. Term " fetal appendage " comprises fetal membrane, consists of the chorion frondosum of the fetal parts of placenta, and comprises Cord blood is at interior umbilical cord. In general, these materials all can directly be obtained behind fetal birth.
The phrase that uses in this specification sheets " essentially no damage method " is to be used to illustrate the method for collecting individual newborn infant or infant's cell, comprising but be not only limited to and use swab or slide directly to scrape the epidermic cell of getting skin histology, or oral cavity, nasal cavity, enteron aisle, vagina, urothelial cell, and corneal cell; Use the venipuncture method to collect peripheral blood cells; Use the percutaneous puncture method to collect epiderm skin cell, dermal cell, myocyte and peripheral nerve sheath theca cell; Use bone (as ilium) puncture method to collect periosteum cell and marrow hemopoietic stem cells; The using-system puncture method is collected thymocyte, chondrocyte, liver cell or splenocyte.The fetal appendage that comprises fetal membrane, placenta and umbilical cord then can directly obtain after the fetus birth, and can use simple ordinary method to separate from these tissues and the required cell of purifying.These cells can comprise hemocyte, the epithelial cell in the amnion tissue and the inoblast in the Cord blood, and syneytiotrophoblast in the chorion frondosum and vascular endothelial cell.
At some in particular cases, also can be born to any growing period of juvenile period (for example 14 years old before), when individuality stands various wounds or operation (amygdala surgical blanking, appendicectomy etc.), in trauma care or surgical procedure, and be unlikely under the precondition that causes tangible additional injuries, also therefrom separate corresponding cell from wound or the required tissue of the local collection of operation.
An important cells source as setting up cell bank of the present invention should should be mentioned that Cord blood here especially.The blood of staying in umbilical cord and the placenta after the fetus birth is usually said Cord blood.Because Cord blood is the same with marrow, wherein contain reproducible " doing " cell, promptly by the initiating cell of other hemocytes such as it white corpuscle of deriving, red corpuscle and thrombocyte, so have crucial medical value.It has been recognized that at present cord blood stem cell is a kind of prematurity immunocyte of uniqueness.In the time will can be used for transplanting (no matter being from body or allotransplantation), these cells are identified as foreign matter and to cause the immune response degree very little.After in the implant patient body, though the hematopoietic disorder that single stem cell also can be caused because of tumor radiotherapy or chemotherapy patient, and the good therapeutic actions of performance such as other and disease of immune system.Because the deficiency in bone marrow stem cell source contains the umbilical cord blood bank that cell " is done " in regeneration equally so if can conventionally set up, and will have more life and obtain treatment.In some cases, Cord blood may be more more effective than marrow.Reason comprises: the possibility that (1) transplants back cord blood cell initiation untoward reaction is littler.For example, when donor and acceptor were the born of the same parents parent, its incidence that causes graft-vs-host reaction (GVHR) was than using low 10 times of marrow; (2) cord blood stem cell is stronger approximately 8～10 times than the propagation and the regenerative power of medullary cell; (3) collect umbilical cord and can not injure mother and baby's body; (4) Cord blood contains the possibility of virus (as HIV, HV) than littler from the marrow of adult donor acquisition; (5) cord blood stem cell more is applicable to gene therapy.
Can use Cord blood to treat multiple inherited disease and hemopathy, and other numerous disease.Can use the disease of Cord blood treatment to comprise malignant hematologic disease (as leukemia, myelomatosis), aplastic anemia, Fanconi anemia, plasmocyte pathology and heredity erythrocyte abnormality etc.At present, the umbilical cord graft mainly is to be used for the treatment of leukemia.According to the present invention, making us interested especially is can be in the mode of umbilical cord blood bank at the medium-term and long-term freezing preservation Cord blood of liquid nitrogen, in order to be used for the treatment of contingent various serious disease Childhood that same individuality grows to even after growing up.In general, when using after two years old, individual self Cord blood is the most effective graft material.Collecting the method for Cord blood and device can be referring to United States Patent (USP) 5,619, and 149,5,916,202 and 5,919,202 etc.
Can use various that set up or just at developing cellular segregation and purification process, under aseptic condition, from organization material, separate and be purified into 0～3 years old newborn infant being used to set up the various dissimilar of cell bank of the present invention or source and/or infant's free cell.These methods for example include but are not limited to enzymolysis partition method (as using trypsinase, elastoser, Collagenase etc.), non-enzymolysis partition method (as sequestrant, glycine and disaccharide etc. such as use EDTA); Density gradient centrifugation based on cell size and density variation; Based on the different cell electrophoresis method of cell surface institute static electrification lotus; Based on the affinity chromatography of cell surface characteristic component (as specificity cell surface antigen, acceptor), and based on cell to special affine adhesive cell separation technology of some material etc.Moreover, also can use specific fluorescence dye (as using fluorescence dye link coupled specific antibody) labeled cell, use then flow cell sorter efficiently and accurately two or more in the isolated cell colony have the cell that different shape and biology characterize.
Separate and the cell of purifying can cultivated through going down to posterity or simply after the propagation, but make " characteristic " cell that cooling freezes preservation.These cells must process before freezing preservation characterized comprise: the cell survival inspection, pollute that inspections, cell type evaluation, isozyme and nuclear cytology analysis, cloning efficiency and specific biological active substance generate and the secreting function evaluation, and Immunological Identification etc.At least after using above-mentioned first three methods to identify, cell is told major part as seed cell, and the cell that takes a morsel uses for current research.The feature that must guarantee all passage cells all is identical or close, promptly preserves 10 years, 20 years, 30 years or the cell after the longer time should be that cell when setting up cell bank is identical or close.Guarantee the stability of cytogene, avoiding the morphological change of clone and aging is the prerequisite condition of setting up cell bank of the present invention.Under the situation in preparation cord blood cell storehouse, generally need only be through can be directly frozen after simple separation and purification and the cell analysis process.The freezing preservation of cells involved and quantitatively recovery with build again, characterized, passage cultivation and the large scale culturing of clone and contain serum or the selection of serum free medium and cultural method, for example can be referring to Animal Cell Cultrue a Practical Approach, Edited by R.Ian Freshney, Oxford University Press, 1992.
The cell bank that the various young somatocyte of collecting in the present invention is based on from newborn infant's (comprising placenta, Cord blood) to infant's body in period is set up, be mainly used in same individuality continue after vital process in the autogenous cell of (comprise teenager after gathering cell or respective organization, grow up and the senium) or tissue transplantation and the new artificial organ and the structure of organ, with repair or the treatment body because of damaged, the various pathological change of tissue that various wound or disease caused or organ and function corresponding disorder, low or forfeiture.In some tumour, anaemia, in the treatment of local organization or organ damage and the reparation, and when being used for lift face and cosmetic purpose, use cord blood stem cell of the present invention storehouse, the bone marrow stem cell storehouse, and the cell bank of other tissue or organ origin, not only avoided the untoward reaction of using other donor seed cell to cause largely even fully, and these cells have all kept the form and the functional character of young cell, thereby work as same individuality and growing up, when middle age and senectitude use these cells, local organization of being bred or rebuilding or organ will keep the gene regulating and the metabolism state of these young cells, the cytobiology behavior is (as propagation and differentiation, cell movement, form is kept, matter transportation, growth and metabolic regulation, cell recognition, cell adhesion be connected, signal representation and transmission, energy exchange, ion is penetrating, antigen presentation etc.), and the special biological active agents of synthetic and release (as extracellular matrix, cytokine and lymphokine, the cell adhesion material, somatomedin, hormone and signal mediator etc.) function.
Remove as the tissue culture that will be used for same individuality future, autogenous cell or tissue transplantation, and outside the cell source of artificial organ in making up, cell bank of the present invention also can be used as nuclear transplantation in the future and somatic gene therapy from body or allosome (as the individual member of the family that is used to have relationship by blood) cell or DNA source.Certainly, take from all kinds cell or its genetic modification product of cell bank of the present invention, also can be used for the various laboratory study purposes in fields such as molecular biology and genetically engineered, cell engineering and organizational engineering.
As previously mentioned, use the cell of cell bank of the present invention to carry out autogenous cell transplantation and Tissue Engineering Study, not only draw materials conveniently, cellular segregation is relative with means of purification and cultural method easy, and does not have any ethics problem.
The following example is intended to describe for example the present invention, rather than limits the await the reply scope of claim of the present invention by any way.
Embodiment
Embodiment 1: based on the preparation from the multiple layer artificial skin transplant of the young cell of body
Present embodiment uses rat animal model, illustrate and use frozen 30 months neonate rat epidermis and dermal cell as seed cell, prepare artificial multiple layer skin, and repair the holostrome skin injury that same animal was caused because of mechanical trauma after old age with the autograft method.
(1) preparation of newborn rat skin cell and cultivation
Select the newborn Wistar rat of healthy term birth and under anesthesia, excise small area holostrome skin from back part of animal.75% alcohol immersion was washed 3 times with D-Hanks liquid after 3 minutes.Skin is cut into 5 * 5mm fritter, and with digestion under neutral protease (40g/L) room temperature 24 hours, mechanical separation epidermis and dermal tissue then.
Face tissue is with containing trypsin 0.25g/L) and the solution of EDTA (0.1g/L) digest after 5 minutes for 37 ℃, digestion product is crossed 200 order mesh screens.With containing penicillin (10
5U/L) and after the DMEM substratum of Streptomycin sulphate (100mg/L) washes 3 times with cell, with the blue staining observation of cell of tongue phenol viability.Then in the DMEM substratum that contains 15% (v/v) foetal calf serum, penicillin and Streptomycin sulphate (concentration is the same), Regular Insulin (80U/L), hydrocortisone (0.8mg/L), Transferrins,iron complexes (5mg/L) and Urogastron (20 μ g/L) and 6.8%CO
2The following 37 ℃ of cultivations of environment.
After dermal tissue digests 4 hours with 37 ℃ of type i collagen enzymes (2.5g/L), basically handle with step according to the method described above, and use contain foetal calf serum (15%, v/v), the substratum of HEPES (10mM) and penicillin and Streptomycin sulphate (concentration is the same), at 5%CO
2The following 37 ℃ of cultivations of environment.
Treat that two kinds of cell proliferations are to enough cell densities (1～5 * 10
6Behind the cell/ml), further carry out cell contamination and morphocytology and identify.Frozen these pure seed cells (shorter as the shelf time, also can be frozen in-70 ℃ of refrigerators) in liquid nitrogen then.In frozen cell liquid, add dimethyl sulfoxide (DMSO) (DMSO) and glycerine as protective material, and select suitable freezing and freeze thawing speed.Used cell freezing liquid is for having added the above-mentioned substratum of 10% (v/v) DMSO in the present embodiment, and chilling rate is-1 ℃/minute.During use, freeze pipe is taken out from liquid nitrogen and directly puts into 37 ℃ of water baths and vibrate, melt fully up to content.Low-speed centrifugal and add fresh culture after, continue culturing cell according to a conventional method.
The NIH3T3 cell that uses mitomycin (4mg/L) processing is inoculated epithelial cells (2.0 * 10 as the trophocyte
4/ cm
2) back in 37 ℃ and 6.8%CO
2Condition is cultivated it.Change a subculture in per 3 days, cover with cell monolayer after about 10 days.Repeatedly with containing EDTA (0.1g/L) and trypsin 0.25g/L) the simple digestion of solution after, the continuation culturing cell also goes down to posterity.In addition, the inoblast suspension of cultivating is pressed 1.0 * 10
5/ cm
2Density be inoculated in the glass culturing bottle, cultivate by the ordinary method of above-mentioned cultivation epithelium epithelial cells basically and go down to posterity.
Use tetrazolium bromide (MTT) dyestuff, with survival rate and definite growth curve of normal dyeing exclusive method or two kinds of skin cellss of colony forming efficiency assay method detection by quantitative.Cultured cells well-grown (referring to attached Fig. 1 and 2) after as seen morphological examination recovers.
(2) preparation of matrix rack and multiple layer artificial skin
In order to prepare the matrix rack of multiple layer artificial skin, 2% (w/v) chitin solution that in 2.0% acetic acid solution, make with from the collagen protein of mouse skin tissue extraction mixed by 1: 2 (v/v), and add a small amount of hyaluronic acid (1mg/L), transforming growth factor-alpha
1(TGF α
1, 10 μ g/L) and fibronectin 0.1% (w/v).Be stirred to spumescence fast with under 4 ℃ in the mixture, casting mold and lyophilize then obtains the spongy artificial skin matrix rack that thickness is about 1.5mm.Press present embodiment method preparation artificial skin matrix rack the ultramicroscopic morphology structure as shown in Figure 3.
The neonate rat inoblast of going down to posterity is cultivated by 3.0 * 10 in recovery back
5Cell/cm
2Density be inoculated in a side of above-mentioned matrix rack, cultivate 5 days (middle change nutrient solution 1 time) for 37 ℃.To press 4.0 * 10 through the epithelial cells of above-mentioned same processing then
5Cell/cm
2Density be inoculated in the opposite side of this matrix rack.Place the nutrient solution of above-mentioned cultivation epithelial cells to continue for 37 ℃ to cultivate 5 big (nutrient solution is changed 2 times in the centre) the matrix rack of having inoculated two skin cellss, and then use gas-liquid face culture method instead and cultivate.After cultivation is finished, can will answer the wound site of the damaged same senile rat of layer artificial skin transplant implantation skin and sub-dermal soft tissue.
Preparation is the multiple layer artificial skin of basic comprising cell from body newborn skin cell with rat as stated above, morphological examination visible cell level increases with the prolongation of incubation time, and grows, is divided into well-bedded basal layer cell, acantholysis cell, granular layer cell and flat keratinocyte gradually.Simultaneously, there is desmosome to connect between visible cell, and as seen tonofilament arranged in the cell and be in the keratohyalin granule and the membrane-coating granules of different developmental phases.In addition, along with the prolongation gradually of incubation time, visible lower floor inoblast is increased gradually, and gos deep between epidermis and rack to epidermis.Simultaneously, rack is degraded gradually, and is produced by inoblast and extracellular matrix that a large amount of collegen filament, spandex and the related protein composition that discharge are formed replaces.
(3) observe based on transplantation effect from the multiple layer artificial skin of body neonatal cell
To grow to the senium (about 30 monthly ages), provide when newborn the holostrome skin samples and cause the animal model of the same Wistar rat of holostrome skin wound as present embodiment in the another location, back.After the wound 2 hours, implant the multiple layer artificial skin of as above preparation to inoculate the mode that a fibroblastic side presses close to the surface of a wound.Transplanting the back observed and analyzed by gross examination of skeletal muscle, light microscopic, Electronic Speculum and infrared thermal imagery scan method every 3 days based on the multiple layer artificial skin of the autologous skin cell repairing effect to the geriatric animals skin wound.The result as seen, 3 days grafts of post-transplantation and normal skin tissue's good knitting, and closely with wound-surface plaster.As seen a small amount of capillary vessel graft of growing into was arranged under the mirror after 6 days, and the newborn skin of the graft of growing into is with the normal skin color is very close on every side.Postoperative 9 days, as seen graft zone capillary vessel rolls up, the high-visible well-bedded stratum basale of epidermal area, stratum spinosum epidermidis, granular layer and stratum corneum, and as seen the top layer has keratose to come off, simultaneously visible skin corium cell number and extracellular matrix roll up (referring to Figure 4 and 5).The 15th day visible surface of a wound of postoperative heals substantially, and it is normal to repair district's skin color, and residual scar few.
Claims (10)
1. the autogenous cell storehouse that comprises the cell of preserving by different tissues or organ origin separation, purifying and freeze-drying.
2. according to the cell bank of claim 1, wherein the seed cell of cell bank comprises epidermis epithelial cells, epithelial cell, inoblast, mesenchymal cell, breaks up completely or undifferentiated peripheral blood cells, marrow hemopoietic stem cells, periosteum cell, chondrocyte, cord blood stem cell, chondrocyte, myocyte, immune system cell, epilemma cell, syneytiotrophoblast and hair cell vascular endothelial cell.
3. according to the cell bank of claim 1, the seed cell in the wherein said cell bank derives from newborn infant or its appurtenant, and the infant.
4. according to the cell bank of claim 1, wherein said cell bank seed cell is an autogenous cell.
5. according to the cell bank of claim 1, the seed cell in the wherein said cell bank through or do not pass through genetic modification.
6. according to the cell bank of claim 1, the seed cell in the wherein said cell bank obtains by the not damaged method basically.
7. according to the cell bank of claim 1, wherein said cell bank was preserved 30 years at least.
8. after growing up or be old, same individuality carries out application in Transplanted cells or the tissue repair according to the respective organization cell of the cell bank of each qualification in the claim 1 to 7.
9. application according to Claim 8, wherein said application comprises the host cell that is used for as the gene therapy foreign DNA.
10. application according to Claim 8, wherein said application comprise and are used for cosmetic surgery.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3102124A1 (en) * | 2014-02-06 | 2016-12-14 | Stryker European Holdings I, LLC | Bone marrow harvesting and storage |
| WO2018010588A1 (en) * | 2016-07-13 | 2018-01-18 | 博雅干细胞科技有限公司 | Therapeutic agent for restoring body function aging and delaying organ function decline |
| US10231743B2 (en) | 2015-03-24 | 2019-03-19 | Stryker European Holdings I, Llc | Bone marrow harvesting device and storage methods |
-
2002
- 2002-01-16 CN CN02100073A patent/CN1432645A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3102124A1 (en) * | 2014-02-06 | 2016-12-14 | Stryker European Holdings I, LLC | Bone marrow harvesting and storage |
| US10231743B2 (en) | 2015-03-24 | 2019-03-19 | Stryker European Holdings I, Llc | Bone marrow harvesting device and storage methods |
| US11259819B2 (en) | 2015-03-24 | 2022-03-01 | Stryker European Operations Holdings Llc | Bone marrow harvesting device and storage methods |
| WO2018010588A1 (en) * | 2016-07-13 | 2018-01-18 | 博雅干细胞科技有限公司 | Therapeutic agent for restoring body function aging and delaying organ function decline |
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