WO2007069813A1 - Procédés de culture de cellules souches mésenchymales et compositions renfermant des cellules souches mésenchymales destinées à réparer les défauts cutanés - Google Patents

Procédés de culture de cellules souches mésenchymales et compositions renfermant des cellules souches mésenchymales destinées à réparer les défauts cutanés Download PDF

Info

Publication number
WO2007069813A1
WO2007069813A1 PCT/KR2006/002889 KR2006002889W WO2007069813A1 WO 2007069813 A1 WO2007069813 A1 WO 2007069813A1 KR 2006002889 W KR2006002889 W KR 2006002889W WO 2007069813 A1 WO2007069813 A1 WO 2007069813A1
Authority
WO
WIPO (PCT)
Prior art keywords
mesenchymal stem
pro
stem cells
adipose tissue
fibroblast
Prior art date
Application number
PCT/KR2006/002889
Other languages
English (en)
Inventor
Yun Young Kim
Chan Woong Park
Original Assignee
Modern Cell & Tissue Technologies Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Modern Cell & Tissue Technologies Inc. filed Critical Modern Cell & Tissue Technologies Inc.
Priority to KR1020087014489A priority Critical patent/KR101083047B1/ko
Publication of WO2007069813A1 publication Critical patent/WO2007069813A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components

Definitions

  • the present invention relates to mesenchymal stem cells, in particular, to methods for culturing adipose tissue-derived mesenchymal stem cells and compositions for repairing skin defects comprising mesenchymal stem cells.
  • adipose tissue in which autologous adipose tissue is obtained from a patient oneself by liposuction and transplanted into dermis for improving skin wrinkle has been done since the 1980's. It has been fully verified in terms of safety and therefore became prevailing therapy.
  • liposuction induces wounding of adipose tissue and adipose tissue comprising contaminants such as blood transplanted shows low attachment rate and is readily absorbed, resulting in short period effect (several months) in removal of skin wrinkle. Therefore, re-operation is generally required (Chajchir A., Plast. Reconstr. Surg. 84(6):921- 935(1989); 0. Onur Erol 00, Plast.
  • the present inventors have made intensive researches to develop a novel cell culturing protocol for obtaining mesenchymal stem cells highly expressing collagen and exhibiting practical therapeutic efficacies to skin defects in clinical applications.
  • a two-step culturing protocol comprising an initial culture in a serum-containing medium and a final culture in a serum-free medium gives rise to the production of mesenchymal stem cells highly expressing collagen.
  • a method for culturing a mesenchymal stem cell highly expressing collagen which comprises the steps of: (a) culturing a mesenchymal stem cell isolated from an adipose tissue in a serum- containing medium; and (b) culturing the mesenchymal stem cell in a serum-free medium, wherein the expression level of type I pro-collagen of the mesenchymal stem cell cultured in the step (b) is higher than that of fibroblast and the expression level of pro-MMP-1 (pro-matrix metalloprotease-1) of the mesenchymal stem cell cultured in the step (b) is lower than that of fibroblast.
  • pro-MMP-1 pro-matrix metalloprotease-1
  • the present inventors have made intensive researches to develop a novel cell culturing protocol for obtaining mesenchymal stem cells highly expressing collagen and exhibiting practical therapeutic efficacies to skin defects in clinical applications.
  • a two-step culturing protocol comprising an initial culture in a serum-containing medium and a final culture in a serum-free medium gives rise to the production of mesenchymal stem cells highly expressing collagen.
  • adipose tissues are dissected and treated with suitable proteases, preferably, collagenase.
  • MSC is isolated from the collagenase-treated suspension. Then, MSC isolated is cultured in a serum-containing medium.
  • serum-containing medium means a medium prepared by combining a conventional medium for animal cell and serum.
  • a medium useful in the present invention is any medium used for animal cell culture, for example, including Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)), ⁇ -MEM (Stanner, CP. et al., Nat. New Biol. 230:52(1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147:923(1978)), 199 medium (Morgan et al., Proc. Soc. Exp. Bio.
  • the medium used in the present invention is DMEM, F12, a mixture of DMEM and F12, or RPMI 1640, most preferably, DMEM.
  • the serum used in the step (a) serves as a source to provide growth factors for proliferation of adipose tissue-derived mesenchymal stem cells.
  • the serum useful in this invention is any serum conventionally used in the art, for instance, including calf serum, fetal bovine serum, horse serum and human serum, preferably serum obtained from patients themselves to be administered with mesenchymal stem cells.
  • the medium in the step (a) comprises glutamine as a nitrogen source.
  • the medium in the step (a) is DMEM containing serum and glutamine as a nitrogen source. The culturing in the step (a) is carried out for about 16 hr-3 weeks.
  • Adipose tissue-derived mesenchymal stem cells cultured are then in a serum-free medium.
  • Adipose tissue-derived mesenchymal stem cells cultured in the serum-free medium show the increased expression of type I procollagen and the decreased expression of pro-MMP-1 (pro-matrix metalloprotease- 1). Such expression patterns are more evident by comparing with those of fibroblasts.
  • serum-free medium refers to a conventional medium for animal cells not containing serum, preferably, a growth factors-free medium.
  • a medium useful in the step (b) is any medium used for animal cell culture, preferably, DMEM, F12, a mixture of DMEM and F12, or RPMI 1640, more preferably, a mixture of DMEM and F12, most preferably, a mixture of DMEM and F12 (3:1).
  • the period of time for culturing in the step (b) is shorter than that of the step (a).
  • Long-term culture in the step (b) is responsible for lack of nutrients for cell growth and division.
  • the period of time for culturing in the step (b) is generally 10 min-24 hr, preferably 30 min-12 hr, more preferably 30 min-6 hr, most preferably 30 min- 3 hr.
  • Adipose tissue-derived mesenchymal stem cells are differentiated into adipocyte in more efficient manner than other mesenchymal stem cells. In addition, the ossification of adipose tissue-derived mesenchymal stem cells occurs at lower rate (Gerard Ailhaud, Biochemi and Biophys Res Commun. 315(2):255-263(2004); Sakaguchi Y., Arthritis Rheum. 52(8)2521-9(2005)).
  • Mesenchymal stem cells cultured according to this invention show the increased expression of type I procollagen and the decreased expression of pro-MMP-1 (pro-matrix metalloprotease-1) compared with those of fibroblasts, thereby giving rise to significantly enhanced collagen production capacity.
  • mesenchymal stem cells cultured according to this invention are transplanted for repairing skin defects, they exhibit treatment efficacies to skin defects compared with fibroblasts. Considering the fact that collagen in skin is mainly produced by fibroblasts, the accomplishments of this invention appear surprising.
  • the present invention provides for the first time in vitro cultured adipose tissue-derived mesenchymal stem cells having practical potential to repair skin defects.
  • a pharmaceutical composition for repairing a skin defect which comprises (a) a pharmaceutically effective amount of adipose tissue-derived mesenchymal stem cells; and (b) a pharmaceutically acceptable carrier.
  • a method for repairing a skin defect which comprises administering to a subject a pharmaceutical composition comprising (a) a pharmaceutically effective amount of adipose tissue- derived mesenchymal stem cells; and (b) a pharmaceutically acceptable carrier.
  • adipose tissue- derived mesenchymal stem cells for manufacturing a medicament for treating pigmentation disorders.
  • repairing skin defects refers to the treatment of skin defects associated with deficiency or defect of dermis or subcutaneous tissues, including removal of skin wrinkle, reforming the contour of face, regeneration of collapsed regions due to wound or malformation, treatment of scars due to acne, correction of lip hypoplasia and correction of stretch mark.
  • the adipose tissue-derived mesenchymal stem cells used in this invention are prepared by the present method described hereinabove.
  • the composition of this invention may comprise other cell types ⁇ e.g., fibroblasts or adipocytes) as well as mesenchymal stem cells.
  • the composition is free from other human-originated cells than mesenchymal stem cells, i.e., comprising only mesenchymal stem cells as active ingredients.
  • the mesenchymal stem cells used in the composition express type I pro-collagen in higher level than fibroblast and express pro-MMP-1 (pro-matrix metalloprotease-1) in lower level than fibroblast.
  • the expression level of type I pro-collagen in the mesenchymal stem cells used in the composition is 1.5-7 folds, preferably, 2-6 folds higher than that of fibroblasts.
  • the expression level of pro-MMP-1 in the mesenchymal stem cells used in the composition is 3-130 folds, preferably 4-125 folds lower than that of fibroblasts.
  • the mesenchymal stem cells used in the composition may be autologous or allogenic to subjects to be administered.
  • the autologous mesenchymal stem cells are utilized in the composition, which is prepared by isolating and culturing mesenchymal stem cells from adipose tissues of a patient oneself.
  • the pharmaceutical composition of this invention comprises a pharmaceutically acceptable carrier as well as adipose tissue-derived mesenchymal stem cells.
  • the pharmaceutically acceptable carrier which is commonly used in pharmaceutical formulations, but is not limited to, includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, rubber arable, potassium phosphate, arginate, gelatin, potassium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and mineral oils.
  • the pharmaceutical composition according to the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, and a preservative.
  • the pharmaceutical composition may further comprise other agents for removing skin wrinkle, e.g., botox, Restylane, fibrinogen or collagen. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th ed., 1995), which is incorporated herein by reference.
  • the pharmaceutical composition according to the present invention is intended to be transplanted into skin.
  • a suitable dosage amount of the pharmaceutical composition of the present invention may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, pathogenic state, diet, administration time, administration route, an excretion rate and sensitivity for a used pharmaceutical composition.
  • pharmaceutically effective amount refers to the amount sufficient to exhibit therapeutic efficacies to skin defects described above.
  • the pharmaceutical composition of the present invention may be administered with a daily dosage of 10 4 -10 8 cells.
  • the pharmaceutical composition according to the present invention may be formulated with pharmaceutically acceptable carrier and/or vehicle as described above, finally providing several forms a unit dose form and a multi-dose form.
  • the formulations include, but not limited to, a solution, a suspension or an emulsion in oil or aqueous medium, an extract, an elixir, a powder, a granule, a tablet and a capsule, and may further comprise a dispersion agent or a stabilizer.
  • the pharmaceutical composition of this invention may be formulated in the form of film.
  • melanocytes and keratinocytes, or melanocytes and fibroblasts isolated from normal skin tissues are cocultured in one sheet of film and then the film sheet is applied to affected regions.
  • the formulation of the pharmaceutical composition preferably, is in the forma of injections for dermal or subcutaneous injection.
  • the pharmaceutical composition of this invention is a cell therapeutic to successfully ameliorate or treat skin defects. Where autologous mesenchymal stem cells, the pharmaceutical composition of this invention is completely free from immunological adverse effects.
  • Figs. Ia and Ib represent the results of FACS (fluorescence-activated ceil sorting) analysis for mesenchymal stem cells isolated in Example.
  • Fig. 2 shows the results of Oil Red O staining for verifying differentiation potential of mesenchymal stem cells of this invention to adipocytes.
  • Fig. 3 shows the results of Von kossa's staining and Alizarin red staining for verifying differentiation potential of mesenchymal stem cells of this invention to osteoblasts.
  • Fig. 4 shows the results of Safranin O staining and type II collagen staining for verifying differentiation potential of mesenchymal stem cells of this invention to chondrocytes.
  • Fig. 5a is a graph demonstrating the expression pattern of type I pro-collagen of mesenchymal stem cells of this invention.
  • Fig. 5b is a graph showing the expression pattern of pro-MMP (matrix metalloprotease)-l of mesenchymal stem cells of this invention.
  • Fig. 6a is an image demonstrating the expression pattern of vimentin of mesenchymal stem cells of this invention transplanted into nude mice.
  • Fig. 6b is an image demonstrating the expression pattern of PCNA (proliferating cell nuclear antigen) of mesenchymal stem cells of this invention transplanted into nude mice.
  • Fig. 7 is an image demonstrating the expression pattern of type I collagen of mesenchymal stem cells of this invention transplanted into nude mice.
  • EXAMPLES EXAMPLE 1 Isolation and Culture of MSC (mesenchymal stem cell) from Adipose Tissues from Patients
  • Adipose tissues obtained by liposuction or surgical operation were washed ten times using PBS containing 10% penicillin-streptomycin to remove blood and other contaminants and dissected into 0.2-0.3 g sections.
  • the adipose tissue sections were incubated in 37°C-water bath (100 rpm) for 1 hr.
  • the tissues were treated with collagenase and filtered into 100 ⁇ m mesh to separate a solution portion containing digested components from debris, followed by adding the same volume of FBS to the separated solution portion.
  • the solution was centrifuged at 4 0 C and at 1200 rpm and the supernatant containing lipids and fat layer was removed.
  • MSCGM Mesenchymal stem cell growth media: MSC minimal medium (Cambrex, Walkersville, MD, USA), mesenchymal cell growth supplements (Cambrex, Walkersville, MD, USA), 4 mM L- glutamine and penicillin (0.025 unit/500 ml)/streptomycin (0.025 mg/500 ml)] was added to precipitated MSC and centrifuged at 4 0 C, 1200 rpm for 5 min.
  • MSCGM is a DMEM (Dulbecco's modified Eagle's medium)-based medium containing bovine fetal bovine serum. Then, the supernatant was discarded and MSC prepared thus was inoculated into culture dishes, followed by culturing in 5% CO 2 incubator at 37 0 C. The medium was changed every second day.
  • MSC cells isolated from two patients were cultured for three passages and their aliquots (5 x 10 5 cells) were incubated for 30 min on ice with the primary antibody, anti-CD34 antibody, anti-CD29 antibody, anti-CD44 antibody and anti- CD105 antibody (Dako, Carpinteria, CA, USA) diluted (1:50) in the FACS solution (Bacton Dickinson, San Jose, CA, USA). MSC cells were then washed twice with the FACS solution, centrifuged and incubated for 30 min on ice with the FITC-conjugated secondary antibody (Vector, Burlingame, CA, USA) diluted (1:100) in the FACS solution.
  • the primary antibody anti-CD34 antibody, anti-CD29 antibody, anti-CD44 antibody and anti- CD105 antibody
  • MSC cells were washed once with the FACS solution, they were centrifuged and suspended in 0.5 ml of the FACS solution. Finally, FACS analysis was performed using a FACS apparatus (FACSort, Barton Dickinson, San Jose, CA, USA). As a negative control, MSC cells incubated only with the FITC-conjugated secondary antibody were used.
  • the MSC set (Al-MSC) isolated from adipose tissues was revealed to be negative to a hematopoietic stem cell marker, CD34 and positive to a endogrin receptor, CD105 and CD44, and ⁇ -1-integrin 29, demonstrating the MSC set isolated exhibits MSC characteristics.
  • another MSC set (A4-MSC) isolated from adipose tissues was also revealed to be negative to a hematopoietic stem cell marker, CD34 and positive to a endogrin receptor, CD105 and CD44, and ⁇ -1-integrin 29, demonstrating the MSC set isolated exhibits MSC characteristics.
  • the cells Upon reaching 100% confluency, the cells were cultured for 3 days in an induction medium for differentiation to adipocytes [adipocyte induction medium, human insulin, L- glutamine, MCGS (mesenchymal stem cell supplement), dexamethasone, indomethaci, IBMX(3-isobutyl-l-methyl-xanthine) and penicillin/streptomycin: Cambrex, Walkersville, MD, USA)] without changing a medium and then cultured for 1-3 days in a maintenance medium (adipocyte maintenance medium, human insulin, L-glutamine, MCGS, penicillin/streptomycin: Cambrex, Walkersville, MD, USA) without changing a medium. The induction/maintenance culturing for differentiation to adipocytes was repeated three times. Finally, the cells were cultured for 7 days in the maintenance medium.
  • adipocyte induction medium human insulin, L- glutamine, MCGS (mesenchymal stem cell supplement), dexamethasone, in
  • the cells differentiated into adipocytes were fixed in 10% formalin solution for 20 min at room temperature and rinsed three times with PBS to remove formalin.
  • O stock solution (Sigma, Saint Louis, MO, USA) dissolved in isopropyl alcohol for 15 min and washed sequentially with 30% isopropyl alcohol and distilled water.
  • the cells were counterstained with Harris hematoxylin for 1 min at room temperature and washed with distilled water, followed by mounting.
  • MSC isolated according to this invention has a differentiation potential to adipocytes.
  • the cells differentiated into osteoblasts were fixed in 10% formalin solution for 20 min at room temperature and rinsed three times with DPBS (without calcium PBS) to remove formalin. After washing once with distilled water, the cells were incubated in 5% silver nitrate solution distilled in DW for 1 hr at room temperature. During the incubation, a foil was placed at the bottom to ensure the reaction to be maximally exposed to light. The light of more than 60 W was used. The cells were washed three times with DW, subjected to neutralization with 5% hypo(sodium thiosulphate) for 5 min and washed once with DW, followed by counterstaining with Nuclear-fast Red (Sigma, Saint Louis, MO, USA) for 5 min. The cells stained were washed with DW and mounted.
  • MSC isolated according to this invention has a differentiation potential to osteoblasts.
  • EXAMPLE 7 Alizarin Red Staining
  • the cells differentiated into osteoblasts were fixed in 10% formalin solution for 20 min at room temperature and rinsed three times with DPBS to remove formalin. After washing once with DW, the cells were incubated in 2% alizarin red S solution (Sigma, Saint Louis, MO 7 USA) distilled in DW for 5min at room temperature and then in 90% acetone for 30 sec, followed by drying.
  • alizarin red S solution Sigma, Saint Louis, MO 7 USA
  • the Alizarin Red staining of cells induced to differentiate to osteoblasts from MSC showed osteoblasts stained and Ca 2+ precipitated. Longer induction time for differentiation gave rise to generation of more osteoblasts and precipitated Ca 2+ . Therefore, it could be recognized that MSC isolated according to this invention has a differentiation potential to osteoblasts.
  • the cell suspension was centrifuged at 150 x g for 5 min and cell pellet formed was cultured in 5% CO 2 incubator at 37 0 C.
  • the medium was changed with 0.5 ml of fresh complete chondrogenesis medium every other day and the culturing was performed for 14-28 days.
  • the cells differentiated into chondrocytes were fixed in 10% formalin solution for 1 hr at room temperature and paraffinized, followed by dissecting into 6 ⁇ m sections.
  • the paraffin sections were incubated sequentially in xylene three times for 5 min, 100% ethanol three times for 2 min, 90% ethanol once for 1 min, 80% ethanol once for 1 min and 70% ethanol once for 1 min, and then washed with PBS for 10 min.
  • the cells were stained with Harris hematoxylin for 4 min and washed with DW to remove residual staining solution. Afterwards, the cells were stained with 0.02% Fast Green FCF (Sigma, Saint Louis, MO, USA) for 3 min and incubated in 1% acetic acid for 30 sec.
  • the cells were stained with 0.1% Safranin-0 (Sigma, Saint Louis, MO, USA) for 5 min and incubated sequentially in 70% ethanol ten times for 1 sec, 80% ethanol ten times for 1 sec, 90% ethanol ten times for 1 sec, 100% 80% twice for 1 min and xylene three times for 3 min, followed by mounting with a mounting solution.
  • the Safranin-0 staining of cells induced to differentiate to chondrocytes from MSC showed chondrocytes stained. Longer induction time for differentiation gave rise to generation of more chondrocytes.
  • 3 weeks and 4 weeks are a period of time for differentiation.
  • EXAMPLE 10 Immunohistochemical Staining to Type II Collagen
  • a paraffin block of cells differentiated into chondrocytes were dissected into paraffin sections of 4 ⁇ m thickness and dried.
  • the paraffin sections were incubated sequentially in xylene three times for 5 min, 100% ethanol three times for 2 min, 90% ethanol once for 1 min, 80% ethanol once for 1 min and 70% ethanol once for 1 min, and then washed with PBS for 10 min.
  • the cells deparaffinized were incubated in 0.5% H 2 O 2 solution in methanol for 10 min, 0.3% PBST for 5 min and finally 0.01 M sodium citrate solution heated to 100 0 C for 10 min.
  • the cells were incubated in 1% normal blocking serum-containing PBS for 30 min for preventing nonspecific protein binding.
  • the cells were incubated for 40 min with a primary antibody anti-type II collagen antibody (Dako, Carpinteria, CA, USA) contained in 1% normal blocking serum.
  • the cells were incubated with biotin-conjugated universal secondary antibody (Universal Secondary Antibody, Vector, Burlingame, CA, USA) for 35 min and reacted with streptavidin-horseradish peroxidase (Vector, Burlingame, CA, USA) for 30 min.
  • the color development was induced using 3.5% H 2 O 2 and nickel contained DAB solution.
  • the counterstaining was performed using Fast Red.
  • the cells were incubated sequentially in 70% ethanol ten times for 1 sec, 80% ethanol ten times for 1 sec, 90% ethanol ten times for 1 sec, 100% 80% twice for 1 min and xylene three times for 3 min and mounted with a mounting solution.
  • MSC isolated according to this invention has a differentiation potential to chondrocytes.
  • EXAMPLE 11 MSC Culture under Serum Free Culture Conditions MSC cells isolated in Example 1 were cultured under serum-free ⁇ i.e., growth factor-free) conditions. MSC isolated in Example 1 was seeded at a density of 1.6 x 10 5 cells into 6-well plates containing MSCGM [mesenchymal stem cell growth media: MSC minimal medium (Cambrex, Walkersville, MD, USA), mesenchymal cell growth supplements (Cambrex, Walkersville, MD, USA), 4 mM L-glutamine and penicillin (0.025 unit/500 ml)/streptomydn (0.025 mg/500 ml)] and cultured for 18 hr in 5% CO 2 incubator at 37 0 C.
  • MSCGM meenchymal stem cell growth media: MSC minimal medium (Cambrex, Walkersville, MD, USA), mesenchymal cell growth supplements (Cambrex, Walkersville, MD, USA), 4 mM L-glutamine and penicillin (0.025 unit/500 m
  • FGM-2 (Cambrex, Walkersville, MD, USA) was used. Then, the medium was changed with a serum-free ⁇ i.e., growth factor-free) medium, p-medium (mixed medium of DMEM and F12, 3:1) and cells were cultured for 6 hr in 5% CO 2 incubator at 37 0 C.
  • OD values are represented as mean ⁇ SD.
  • NF46 denotes fibroblast and MSC samples isolated from three patients were utilized.
  • OD values are represented as mean ⁇ SD.
  • NF46 denotes fibroblast and MSC samples isolated from three patients were utilized.
  • the expression level of type I procollagen in MSC of this invention is significantly increased compared with that of fibroblasts.
  • the elevated expression level of type I procollagen in MSC of this invention is a surprising result, demonstrating that MSC of this invention itself or together with fibroblasts can be used as a promising cell therapy for removing skin wrinkle.
  • MSC of this invention has a highly decreased activity to collgen degradation, which contributes to the increase in efficacies of MSC of this invention to remove skin wrinkle.
  • a transplantation site was marked using a 10 mm biopsy punch stained with a staining solution.
  • 0.5-1 x 10 6 cells of MSC isolated and cultured in Example 1 were suspended in 150 ⁇ l of 5% glucose physiological saline and injected into the marked transplantation site using a 1 ml syringe.
  • For a negative control only 5% glucose physiological saline was injected.
  • dermis and subcutaneous skin tissues at the transplantation site were dissected using a 10 mm biopsy punch and fixed in 10% formalin. The fixed tissues were paraffin-sectioned and subjected to immunostaining by use of anti-vimentin antibody (Dako, Carpinteria, CA, USA) and anti-PCNA antibody (Dako, Carpinteria, CA, USA).
  • a transplantation site was marked using a 10 mm biopsy punch stained with a staining solution.
  • MSC in Example 1 were suspended in 150 ⁇ l of 5% glucose physiological saline and injected into the marked transplantation site using a 1 ml syringe. For a negative control, only 5% glucose physiological saline was injected. After 7-28 days of injection, dermis and subcutaneous skin tissues at the transplantation site were dissected using a 10 mm biopsy punch and frozen-sectioned, followed by fluorescence immunostaining by use of anti-collagen type I antibody (Chemichon, Temecula, CA, USA) and anti-BrdU antibody (Dako, Carpinteria, CA, USA).
  • the transplanted cells could be traced by fluorescence immunostaining using anti-BrdU antibody after 7 and 14 days of transplantation. It could be observed by fluorescence immunostaining using anti-collagen type I antibody that type I collagen was also expressed in cells positive to BrdU. Accordingly, it could be appreciated that MSC injected expresses type I collagen normally in dermis of nude mouse, leading us to conclude that mesenchymal stem cells of this invention are excellent cell therapeutics to repair dermis defects such as skin aging, wrinkle and collapse associated with collagen damage and degradation in dermis layer.
  • the present invention provides a method for culturing a mesenchymal stem cell highly expressing collagen. Furthermore, the present invention provides a pharmaceutical composition for repairing a skin defect. According to the present invention, it is possible to obtain adipose tissue-derived mesenchymal stem cells having higher expression level of type I pro-collagen and lower expression level of pro-MMP-1.
  • the pharmaceutical composition of this invention is a cell therapeutic to successfully ameliorate or treat skin defects. Where autologous mesenchymal stem cells, the pharmaceutical composition of this invention is completely free from immunological adverse effects.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Rheumatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de culture de cellules souches mésenchymales à haute expression de collagène, qui consiste à: (a) mettre en culture une cellule souche mésenchymale isolée d'un tissu adipeux dans un milieu contenant du sérum; et (b) mettre en culture la cellule souche mésenchymale dans un milieu dépourvu de sérum, le niveau d'expression du procollagène de type I de la cellule souche mise en culture à l'étape (b) étant supérieur à celui du fibroblaste et le niveau d'expression de la pro-MMP-1 (pro-métalloprotéase-1 de la matrice) de la cellules souche mésenchymale mise en culture à l'étape (b) étant inférieur à celui du fibroblaste. En outre, l'invention concerne une composition pharmaceutique de réparation des défauts cutanés qui comprend les cellules souches mésenchymales précitées comme ingrédients actifs.
PCT/KR2006/002889 2005-12-16 2006-07-21 Procédés de culture de cellules souches mésenchymales et compositions renfermant des cellules souches mésenchymales destinées à réparer les défauts cutanés WO2007069813A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020087014489A KR101083047B1 (ko) 2005-12-16 2006-07-21 간엽줄기세포의 배양방법 및 간엽줄기세포를 포함하는 피부결함의 치료용 조성물

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2005-0124320 2005-12-16
KR20050124320 2005-12-16

Publications (1)

Publication Number Publication Date
WO2007069813A1 true WO2007069813A1 (fr) 2007-06-21

Family

ID=38163101

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2006/002889 WO2007069813A1 (fr) 2005-12-16 2006-07-21 Procédés de culture de cellules souches mésenchymales et compositions renfermant des cellules souches mésenchymales destinées à réparer les défauts cutanés

Country Status (2)

Country Link
KR (1) KR101083047B1 (fr)
WO (1) WO2007069813A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2752484A1 (fr) * 2011-08-31 2014-07-09 Sewon Cellontech Co., Ltd Procédé de préparation de milieu de culture basique et milieu de culture basique pour cellules souches mésenchymateuses, et agent thérapeutique cellulaire mis en culture et différencié à l'aide de celui-ci
US10563198B2 (en) 2015-03-11 2020-02-18 Wings Therapeutics, Inc. Oligonucleotides matching COL7A1 exon 73 for epidermolysis bullosa therapy
CN112569343A (zh) * 2020-12-22 2021-03-30 北京唐颐惠康生物医学技术有限公司 一种用于治疗脱发的复合物及其制备方法
JP2021105620A (ja) * 2015-11-24 2021-07-26 オルボテック リミテッド プローブシステム
CN114717185A (zh) * 2022-03-31 2022-07-08 秦岭大熊猫研究中心(陕西省珍稀野生动物救护基地) 一种大熊猫胎盘间充质干细胞分离与培养方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101032271B1 (ko) * 2009-05-25 2011-05-06 (주)미래생명공학연구소 피부세포 재생용 조성물, 그 제조 방법, 피부세포 재생 방법 및 화장료 조성물
JP6867726B1 (ja) * 2020-10-02 2021-05-12 株式会社 バイオミメティクスシンパシーズ 皮膚の健康状態を増進及び／又は改善するための組成物

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003022988A2 (fr) * 2001-09-10 2003-03-20 The Regents Of The University Of California Cellules souches provenant de tissu adipeux et reseaux
WO2003084468A2 (fr) * 2002-04-03 2003-10-16 Artecel Sciences, Inc. Ameliorations apportees a des cellules souches adultes differenciees derivees du tissu adipeux et leurs utilisations
WO2005035738A1 (fr) * 2003-10-07 2005-04-21 Biomaster Inc. Differentiation cellulaire de cellules precurseurs issus des tissus adipeux
WO2005042730A2 (fr) * 2003-11-04 2005-05-12 Biomaster, Inc. Procede et systeme pour preparer des cellules souches a partir de tissus gras

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003022988A2 (fr) * 2001-09-10 2003-03-20 The Regents Of The University Of California Cellules souches provenant de tissu adipeux et reseaux
WO2003084468A2 (fr) * 2002-04-03 2003-10-16 Artecel Sciences, Inc. Ameliorations apportees a des cellules souches adultes differenciees derivees du tissu adipeux et leurs utilisations
WO2005035738A1 (fr) * 2003-10-07 2005-04-21 Biomaster Inc. Differentiation cellulaire de cellules precurseurs issus des tissus adipeux
WO2005042730A2 (fr) * 2003-11-04 2005-05-12 Biomaster, Inc. Procede et systeme pour preparer des cellules souches a partir de tissus gras

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZUK P.A. ET AL.: "Human Adipose Tissue Is a Source of Multipotent Stem Cells", MOLECULAR BIOLOGY OF THE CELL, vol. 13, December 2002 (2002-12-01), pages 4279 - 4295, XP002249630 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2752484A1 (fr) * 2011-08-31 2014-07-09 Sewon Cellontech Co., Ltd Procédé de préparation de milieu de culture basique et milieu de culture basique pour cellules souches mésenchymateuses, et agent thérapeutique cellulaire mis en culture et différencié à l'aide de celui-ci
EP2752484A4 (fr) * 2011-08-31 2015-04-15 Sewon Cellontech Co Ltd Procédé de préparation de milieu de culture basique et milieu de culture basique pour cellules souches mésenchymateuses, et agent thérapeutique cellulaire mis en culture et différencié à l'aide de celui-ci
US10563198B2 (en) 2015-03-11 2020-02-18 Wings Therapeutics, Inc. Oligonucleotides matching COL7A1 exon 73 for epidermolysis bullosa therapy
US11352626B2 (en) 2015-03-11 2022-06-07 Wings Therapeutics, Inc. Oligonucleotides matching COL7A1 exon 73 for epidermolysis bullosa therapy
JP2021105620A (ja) * 2015-11-24 2021-07-26 オルボテック リミテッド プローブシステム
JP7085042B2 (ja) 2015-11-24 2022-06-15 オルボテック リミテッド プローブシステム
CN112569343A (zh) * 2020-12-22 2021-03-30 北京唐颐惠康生物医学技术有限公司 一种用于治疗脱发的复合物及其制备方法
CN114717185A (zh) * 2022-03-31 2022-07-08 秦岭大熊猫研究中心(陕西省珍稀野生动物救护基地) 一种大熊猫胎盘间充质干细胞分离与培养方法

Also Published As

Publication number Publication date
KR20080079256A (ko) 2008-08-29
KR101083047B1 (ko) 2011-11-16

Similar Documents

Publication Publication Date Title
US11369716B2 (en) Reparative cell isolation and delivery
RU2396084C1 (ru) Композиция филлера для мягких тканей для инъекции и способ ее получения
KR100907248B1 (ko) 분화된 어린 지방 세포와 생분해성 중합체의 이식에 의한신체의 부피 대체 방법
Azari et al. Effects of transplanted mesenchymal stem cells isolated from Wharton’s jelly of caprine umbilical cord on cutaneous wound healing; histopathological evaluation
EP2865749A1 (fr) Procédé de production de cellules souches à haute concentration
JP5240715B2 (ja) 脂肪組織由来多分化能幹細胞を含有する細胞製剤
US20120207722A1 (en) Use Of Adipose Tissue Cells For Initiating The Formation Of A Functional Vascular Network
WO2007069813A1 (fr) Procédés de culture de cellules souches mésenchymales et compositions renfermant des cellules souches mésenchymales destinées à réparer les défauts cutanés
Ning et al. Bone marrow mesenchymal stem cells differentiate into urothelial cells and the implications for reconstructing urinary bladder mucosa
WO2010069204A1 (fr) Cellules souches pluripotentes, procédé pour la préparation de celles-ci et utilisations de celles-ci
EP2049130A1 (fr) Composition de charge de tissus mous comportant du produit autologue de culture cellulaire dérivé du derme et de l'acide hyaluronique
TW201103572A (en) Method and composition for restoration of age-related tissue loss in the face or selected areas of the body
Landau et al. proposed methods to improve the survival of adipose tissue in autologous fat grafting
KR101425653B1 (ko) 인간 지방조직 유래 다분화능 줄기세포 및 모낭세포를함유하는 세포치료제
JP5388297B2 (ja) アディポクラスター
Proulx et al. Optimization of culture conditions for porcine corneal endothelial cells
AU2018289935A1 (en) Treatment agent for epidermolysis bullosa
JPWO2006134951A1 (ja) 脂肪由来幹細胞の長期培養増殖法
CN102703380B (zh) 亚全能干细胞、其制备方法及其用途
Zhao et al. Transdifferentiation of autologous bone marrow cells on a collagen-poly (ε-caprolactone) scaffold for tissue engineering in complete lack of native urothelium
EP3950934A1 (fr) Procédé de culture d'une population cellulaire et utilisation correspondante
Alessandri-Bonetti et al. Soft Tissue Reconstruction
EP4144835A1 (fr) Procédé de culture d'une population cellulaire contenant des cellules positives tie2 dérivées du cartilage, et utilisation dudit procédé
KR20190142266A (ko) 항암 바이러스가 도입된 중간엽 줄기세포의 세포 생존능을 향상시키는 방법
CN117305236A (zh) 脂肪间充质干细胞低氧气氛培养及其治疗卵巢早衰的用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: KR

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 06769298

Country of ref document: EP

Kind code of ref document: A1