WO2008091013A1 - 軟骨細胞調製方法 - Google Patents
軟骨細胞調製方法 Download PDFInfo
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- WO2008091013A1 WO2008091013A1 PCT/JP2008/051327 JP2008051327W WO2008091013A1 WO 2008091013 A1 WO2008091013 A1 WO 2008091013A1 JP 2008051327 W JP2008051327 W JP 2008051327W WO 2008091013 A1 WO2008091013 A1 WO 2008091013A1
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
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- 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
- A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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- C12N2501/30—Hormones
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- C12N2501/39—Steroid hormones
Definitions
- the present invention relates to a method for preparing chondrocytes, and more particularly to a method for preparing chondrocytes from perichondrial cells.
- Non-Patent Documents 1 to 4 a method of collecting a part of autologous chondrocytes and culturing them in vitro and then returning them to the affected area is considered (Non-Patent Documents 1 to 4), and clinical applications are also being performed (Non-Patent Documents 5 and 6 and 7).
- chondrocytes has two problems. That is, the problem of invasion to the collection site and the problem of long-term form maintenance.
- the problem of invasion of the first collection site is that when the cartilage used for cartilage culture is collected, the site becomes deformed or defective, such as a defect or a depression.
- the second problem of long-term shape maintenance is whether tissue regenerated with cultured cartilage can continue to maintain its shape without being absorbed for a long time.
- chondrocytes In order to solve these problems, it is considered to seek other sources of chondrocytes.
- the idea is to use cells other than chondrocytes to differentiate into chondrocytes ex vivo and return them to the living body. Examples of these cells include embryonic stem cells, mesenchymal stem cells, knee joint synovial cells, and fat cells (Patent Documents 1 to 4). All have been confirmed to differentiate into chondrocytes.
- Non-Patent Document 1 van Osch GJ et al, Plast Reconstr Surg 107: 433-440 (2001)
- Non-Patent Document 2 Brittberg et al, The New England Journal of Medicine
- Non-Patent Document 3 Ting et al, Annals of Plastic Surgery 40: 413-421 (1998)
- Non-Patent Document 4 Rodriguez et al, Plastic and Reconstruct ive Surgery
- Non-Patent Document 5 0chi M et al, J Bone Joint Surg 84: 571-578 (2002)
- Non-Patent Document 6 Yanaga H et al, Aesth Plast Surg 28: 212-221 (2004)
- Non-Patent Document 7 Yanaga H et al, Plast & Reconstr Surg 117: 2019-30 (2006)
- Non-Patent Document 8 0ve Engkvist et al., Scand ⁇ Plast Reconst Surg. 1979, 13: 275-280
- Non-Patent Document 9 0ve Engkvist et al., Scand J Plast Reconst Surg. 1979, 13: 371-376
- Non-patent literature 10 Duynstee et al., Plasr and Reconst Surg. 2002, 110 (4). 1073-1079
- Patent literature 1 JP 2005 No.-511083
- Patent Document 2 JP 2003-51875 A
- Patent Document 3 Japanese Patent Laid-Open No. 2005-500085
- Patent Document 4 Japanese Patent Laid-Open No. 2001-103965
- An object of the present invention is to provide a method for preparing chondrocytes with less invasion to a collection site.
- the present inventors have developed a method using a perichondrium covering the outer sides of the auricular cartilage and costal cartilage.
- the present inventors have isolated and proliferated perichondrial cells from the perichondrium, and differentiated into chondrocytes in and out of the living body, thereby being a chondrocyte specific substrate. Succeeded in producing oral theoglycan and type II collagen.
- the gist of the present invention is as follows.
- a cell derived from human perichondrial tissue which can differentiate into a chondrocyte
- a composition comprising the cell according to any one of (1) to (3).
- composition according to (5) which is a cell derived from human perichondrial tissue, which is used for growing the cell capable of differentiating into a chondrocyte.
- Cell transplantation is treatment of congenital auricular malformation, treatment of costal cartilage defect, treatment of articular cartilage damage, treatment of tracheal cartilage defect, bulging rhinoplasty, genioplasty, facial concavity (8)
- the composition according to (8) which is intended for any one of facial left / right asymmetry correction, eyelid peripheries, or facial cosmetic surgery.
- Transplantation treatments include congenital auricular malformation, costal cartilage defect treatment, articular cartilage damage treatment, tracheal cartilage defect treatment, nose surgery, genioplasty, facial concavity surgery, (23)
- composition according to any one of 4).
- the method for preparing chondrocytes of the present invention does not require the collection of cartilage tissue, the invasion to the collection site can be minimized.
- FIG. 1 Histological examination at the time of collecting chick perichondrium.
- Cartilage tissue is stained blue in Alcian blue, but the perichondrium is not stained.
- the perichondrium only the outermost layer and the fibroblast layer can be collected, or the outermost layer, the fibroblast layer and the innermost layer (transition between the cartilage matrix) can be collected.
- d Furthermore, the innermost layer of the perichondrium (transition to the cartilage matrix) can also be collected.
- e Perichondrial tissue collected from the outermost layer and fibroblast layer alone Alcian blue staining 200x magnification
- FIG. 2 Centrifugal tube culture of perichondrial cells.
- the perichondrial cell mass forms a cartilage tissue by a centrifuge tube.
- a Cartilage tissue regenerated from perichondrial cells
- b Cartilage tissue regenerated from chondrocytes Alcian blue staining Bar: 200; ani
- peritoneal cells are stratified in the same way as chondrocytes, thereby increasing the ability to produce substrate (pure theorican).
- peritoneal cells are stratified in the same way as chondrocytes, increasing the ability to produce matrix (or theothelican), which is equivalent to that of chondrocytes.
- a Perichondrial cells cultured in a monolayer
- b Perichondrocytes in a multi-layered structure (tri-layered)
- c Monolayered Chondrocytes
- d Multilayered (Trilayered) perichondrial cells Alcian blue staining
- FIG. 5 RT-PCR in a stratified culture of perichondrial cells and chondrocytes.
- type I collagen decreases and type I collagen increases.
- FIG. 6 Quantification by real-time PCR in the stratified culture of perichondrial cells and chondrocytes. As perichondrocytes stratify, type I collagen tends to decrease and type I collagen tends to increase.
- FIG. 8 Cultured human perichondrial cells form cartilage tissue in vivo.
- FIG. 9 In vivo, cultured human perichondrial cells produce type I collagen and type I collagen and form cartilage tissue.
- the chick perichondrium tissue is collected in two layers: the outermost layer and the fibroblast layer and innermost layer. Others become cartilage tissue. a: Outermost layer and fibroblast layer b: Innermost layer c: Cartilage tissue Alcian blue staining Magnification 200 times
- FIG. 12 Proliferative ability of chick shark perichondrial cells. Comparison of colony-forming ability of perichondrocytes and chondrocytes under a microscope.
- Human perichondrocytes formed large colonies after 1 month in culture compared with chick chondrocytes.
- Human chondrocytes (dotted arrows) Human perichondrocytes (solid arrows) Scale bar: 500 um [Fig. 13] Proliferative capacity of human chondrocytes. Baboon perichondrium cells, perichondrium-cartilage transition cells, cartilage Comparison of cell colony forming ability.
- perichondrium When cultured for 14 days using perichondrium, perichondrium-cartilage transition, chondrocytes, and comparing the number of colonies formed from colonies of 50 cells or more, it was found that perichondrocytes have high colony-forming ability. . Therefore, it was found that it has a high proliferation ability limited to the perichondrial cells.
- FIG. 14 Proliferative capacity of chick shark perichondrium cells. Comparison of long-term proliferation ability of chick perichondrium, cartilage-perichondrium transition, and chondrocytes.
- FIG. 16 Induction of cartilage differentiation in vitro.
- A-C, G-I Alcian bull stain
- D-F Type I collagen stain (red), Collagen type collagen stain (green), DAP stain I (blue) Scale bar: 200wn
- FIG. 17 Histological examination of cartilage tissue regenerated from human perichondrium and chondrocytes by in vivo.
- A Human perichondrium 1 month after transplantation, chondrocyte-derived regenerated cartilage (A-D: chondrocyte-derived cartilage tissue, E-H: chondrocyte-derived cartilage tissue)
- Regenerated cartilage derived from chick perichondrial cells was covered with type I collagen, but regenerated cartilage derived from chondrocytes was not covered with type I collagen.
- chondrocyte-derived regenerated cartilage (AD: chondrocyte-derived soft bone tissue, E-H: chondrocyte-derived cartilage tissue)
- the tissue derived from chick perichondrial cells was covered with type I collagen as in the first month after transplantation, but the regenerated cartilage derived from chondrocytes was not covered with type I collagen.
- perichondrocytes regenerate cartilage in the same way as chondrocytes. Regeneration from perichondrial cells Cartilage, unlike regenerated cartilage derived from chondrocytes, is covered with perichondrium. This suggests that regenerated cartilage derived from perichondrial cells is superior in long-term morphology maintenance ability.
- FIG. 18 Number of cells per lmm2 of cartilage reconstituted in vivo.
- the number of cells per lira2 in the cartilage of the tissue removed 1 month and 3 months after transplantation.
- the number of cells did not change at 1 month and 3 months after transplantation, whereas in chondrocyte derived tissue, the number of cells decreased at 3 months after transplantation. It was.
- Perichondrial cells show that long-term morphology maintenance ability is superior to chondrocytes.
- chick perichondrocytes formed large colonies on the 9th day of culture. 40x magnification
- FIG. 20 Comparison of shark perichondrium cells, perichondrium-cartilage transition zone cells, and chondrocytes under a microscope with 103 ⁇ 4 human autologous serum medium.
- cartilage tissue is collected as a lump, treated with an enzyme such as collagenase to isolate chondrocytes from the matrix components, and the isolated chondrocytes are cultured. It has been used for transplantation treatment, especially for autologous transplantation.
- perichondrium and cartilage such as elastic cartilage and hyaline cartilage are formed of four layers (FIG. 10). 1) outermost layer (including capillaries), 2) fibroblast layer (mainly perichondrial cells) (referred to as “perichondrial cell layer” in FIG. 10), 3) innermost layer (with cartilage matrix) ), 4) 4 layers of mature cartilage layer (enclosed by cartilage matrix) (eg, Ba i rat i A, Comazz i M, Gl or ia M. et al., T i ssue Cell 28: 455) —68.
- cartilage matrix eg, Ba i rat i A, Comazz i M, Gl or ia M. et al., T i ssue Cell 28: 455.
- the “outermost layer” includes a capillary that is the uppermost layer of a layer that expresses type I collagen and does not express type I collagen.
- the “fibroblast layer” is a layer that expresses type I collagen and does not express type IV collagen, and is composed entirely of perichondrial cells not including the outermost layer.
- the “innermost layer” includes a “fibroblast layer” and a cartilage matrix that expresses type II collagen and proteodalycan.
- the “mature cartilage layer” is a layer that expresses type I collagen and oral theodarican and does not express type I collagen.
- the cartilage collection method so far is 1)-4) all, or 3) and 4) or 4) only.
- the cartilage tissue at the collection site is missing or does not return to its original shape, and apparently wrinkles such as depressions and deformations may be observed.
- the tissue containing 1) and 2) or 1)-3) may be collected using a sharp instrument such as tweezers and scissors.
- blunt instruments such as a stripper may be used.
- Layers containing chondrocytes that occupy most of the soft tissue need not be collected. Therefore, it is a great advantage that it can be collected with minimal invasion, there is no defect as a cartilage tissue, and no saddle is formed.
- the tissue thus obtained is separated under certain conditions by collagenase or the like (for example, 0.30.33! Collagenase, 37, 1-3 o'clock. This is a separation method peculiar to perichondrial cells.
- 1) -4) can be collected, and when the obtained tissue is separated with collagenase, etc., perichondrocytes and chondrocytes can be removed.
- collagenase was allowed to act.
- the perichondrial cells are separated within 3 hours, but it takes about 10 to 16 hours to separate the chondrocytes, and this time difference can be used to separate the perichondrial cells from the chondrocytes.
- the separated cells are seeded on a culture dish and cultured in a growth medium for about 1 week. Thereafter, perichondrial cells can be differentiated into chondrocytes by performing centrifuge tube culture, monolayer culture or multi-layer culture in a differentiation induction medium.
- human perichondrial cells obtained from human perichondrium excluding human cartilage are cultured. By doing so, perichondrial cells can be differentiated into chondrocytes.
- the present invention provides a cell derived from a perichondrial tissue and capable of differentiating into a chondrocyte.
- the perichondrial tissue-derived cells of the present invention are considered to be cartilage stem and Z or progenitor cells.
- the perichondrial tissue is a part of a tissue constituting the cartilage tissue such as the auricle and the costal cartilage and includes the perichondrium. Specifically, a tissue including an outermost layer and a fibroblast layer (perichondrial cell layer), and a tissue including an outermost layer, a fibroblast layer (perichondrial cell layer) and an innermost layer.
- the human perichondrium tissue from which the cells of the present invention are derived may be composed of an outermost layer and a fibroblast layer, and may be composed of an outermost layer, a fibroblast layer and an innermost layer.
- the perichondrial tissue may be collected from a human, particularly a patient in need of cartilage transplantation.
- the cell derived from the perichondrial tissue may be a cell isolated from the perichondrial tissue or a cell obtained by subculturing the cell.
- perichondrial tissue is collected, and the cells are separated from the collected tissue.
- a sharp instrument such as tweezers and scissors may be used, or a blunt instrument such as an exfoliator may be used.
- the perichondrial tissue is treated with collagenase under certain conditions (for example,
- Du l For primary and subculture of cells isolated from perichondrial tissue, Du l supplemented with serum (eg lOiC Fe B al Bovine Serum (FBS), serum from the patient to be transplanted) becco 's Mod if ied Eagles' s Medium / Nutrient Mixture F 12 (DMEM / F 12) or Nut ri ent Mixture F-12 Ham (F-12 Ham), approx. 37 "C The medium should be changed every 2-4 days.
- serum eg lOiC Fe B al Bovine Serum (FBS)
- FBS lOiC Fe B al Bovine Serum
- F-12 Ham Nut ri ent Mixture F-12 Ham
- Chondrocytes are difficult to cultivate for a long time because they proliferate, but perichondrocytes can be proliferated and cultured for a long time compared to soft bone cells.
- medium containing serum such as DEME / F12, serum (eg 103! FBS, serum from patient to be transplanted)
- antibiotics and Incubate at about 37X: in a medium containing an anti-mitotic agent.
- Antibiotic antimycotic solution SIGMA A5955 etc. can be used as a medicine containing both antibiotics and antimitotics.
- the medium may further contain 40-60 g / ml dexamethasone and 30 or 60-60 g / ml L-ascorbic acid.
- Insulin-like growth factor for example, 5-10 ng / ml Insulinlike growth factor-1 (IGF-1), 5-10 ng / ml basic fibroblast growth factor ( bFGF)), etc.
- IGF-1 Insulinlike growth factor-1
- bFGF basic fibroblast growth factor
- 5-10 ng / ml insulin (Insulin), etc. may be added, and the medium should be changed every 2-3 days.
- Cell clusters can be formed by culturing cells derived from perichondrial tissue in a centrifuge tube. For example, 5ng / ml Insulinlike growth f a'ctor-1 (IGF-1), 5ng / ml basic Fibroblast growth factor (bFGF), 40ng / ml ⁇ Dexamethasone, L-ascorbic acid, Antibiotic ant imicot ic solut ion, Insul in-Transferrin ⁇ Serine (ITS) 3 ⁇ 4 * a In a serum-free medium of DMEM / F12, when cultured for 2 to 4 weeks in a centrifuge tube at about 37 tons, a cell mass is formed.
- IGF-1 Insulinlike growth f a'ctor-1
- bFGF basic Fibroblast growth factor
- ITS Insul in-Transferrin ⁇ Serine
- cells derived from perichondrial tissue can be monolayered or stratified by plate culture.
- DMEM / F12 containing FBS and Antibiotic ant imicotic solut ion or DMEM / F12 10S! FBS l3 ⁇ 4Antibiotic antimicotic solution, 5ng / ml IGF-1, 5ng / ml bFGF, 40ng / ml Dexamethasone
- the medium is plated using a medium containing, and stratified every week, the ability to produce a substrate (eg, proteodarican) increases.
- the number of stratifications varies depending on the application and required tissue size, but usually 3 to 5 times is appropriate.
- composition and content of the medium can be changed as appropriate, and those changes are also within the scope of the present invention.
- the present invention relates to cartilage comprising culturing cells isolated from perichondrial tissue.
- a method for preparing a cell capable of differentiating into a cell is provided.
- the present invention also provides a method for preparing chondrocytes, comprising differentiating cells derived from perichondrial tissue into chondrocytes.
- perichondrial cells and chondrocytes produce different substances. It is known that perichondrocytes are present in type I collagen and chondrocytes are present in type I collagen and proteoglycan. Perichondrial cells and cartilage cells can be distinguished by these products.
- the present invention also provides chondrocytes prepared by differentiating cells derived from perichondrial tissue into chondrocytes.
- chondrocytes derived from perichondrial tissue that can differentiate into chondrocytes into the living body
- treatment of congenital auricular malformations treatment of costal cartilage defects, articular cartilage injury (eg, deformation)
- treatment of osteoarthritis of the knee treatment of tracheal cartilage defect, bulging rhinoplasty, genioplasty, facial indentation, facial asymmetric correction, correction of the area around the eyelids, facial cosmetic surgery, etc.
- substrates produced by these cells for example, proteoglycans such as type I, type II collagen, and aggrecan may be added.
- chondrocytes derived from cartilage tissue may be added.
- the cartilage tissue is preferably collected from a human, particularly a patient in need of cartilage transplantation
- the cartilage tissue is collected and the cells are separated from the collected tissue.
- the cartilage tissue should be treated with collagenase under certain conditions (eg, 0.1-0.23 ⁇ 4! Collagenase, 37t: 10-16 hours).
- collagenase under certain conditions (eg, 0.1-0.23 ⁇ 4! Collagenase, 37t: 10-16 hours).
- the number of cells required for transplantation can be prepared in a shorter time because the number of cells in the primary culture is high. it can. Or during planting is implanted without adding anything, I, or the like may be added I I collagen or Puroteodarikan.
- Cell transplantation can be performed by injecting the cells derived from the perichondrial tissue and capable of differentiating into chondrocytes into the affected area with a syringe.
- Cells derived from the perichondrial tissue which can be differentiated into chondrocytes, are cultured with a scaffold (soft bone treatment material) to form a three-dimensional structure of cartilage tissue.
- a scaffold soft bone treatment material
- the scaffold material include collagen, polylactic acid, polyglycolic acid, a copolymer of polylactic acid and polyglycolic acid, and a non-absorbable material such as polyethylene.
- treatment of congenital auricular malformation treatment of costal cartilage defect using soft bone cells prepared by differentiating cells derived from perichondrial tissue into chondrocytes and Z or cartilage tissue formed by these chondrocytes .
- Treatment of articular cartilage damage eg osteoarthritis of the knee
- treatment of tracheal cartilage defect eg bulging rhinoplasty, genioplasty, facial indentation, facial asymmetry correction, correction around the eyelid
- a substrate produced by these chondrocytes for example, I, I type I collagen, proteodarican, aggrecan
- a substrate produced by these chondrocytes for example, I, I type I collagen, proteodarican, aggrecan
- cartilage cells derived from cartilage tissue may be added.
- the method for obtaining chondrocytes and the advantages of adding chondrocytes are as described above.
- perichondrial cells can be placed in the material by placing an absorbable or non-absorbable material at the bottom of the incubator during cell culture. It can be transplanted directly to the affected area.
- Chondrocytes can be transplanted by injecting the cells into the affected area with a syringe.
- Transplantation of cartilage tissue using a material scaffold can be performed by surgical transplantation.
- surgical techniques such as bulging rhinoplasty and auricular plastic surgery can be used.
- the present invention provides a method for preparing a matrix produced by chondrocytes, comprising differentiating perichondrial cells into chondrocytes, and causing the chondrocytes to produce a matrix.
- the substrate produced by chondrocytes include II type I collagen and proteodalycan. These substrates can be used for cosmetics, foods, health foods, pharmaceuticals, and the like.
- the perichondrial tissue at the time of collection refers histologically to the outermost layer and the fibroblast layer, and the perichondrium-cartilage transition tissue refers to the innermost layer.
- Cartilage tissue such as auricles and costal cartilage (using perichondrium obtained from surplus human auricular cartilage at the time of surgery with the consent of the person or parents.
- Kanagawa Children's Medical Center and Yokohama City University School of Medicine Sharp instruments such as tweezers and scissors were used to collect perichondrial tissue from the ethics committee of the attached hospital.
- a blunt instrument such as a stripper may be used.
- a tissue section Fig. 1
- the collected perichondrial tissue was confirmed for each individual by Alcian blue staining, which is a staining specific to the cartilage matrix.
- the collected perichondrial tissue was not stained at all by Alcian blue staining, but part of the perichondrium-cartilage transition tissue and all of the cartilage tissue were stained (Fig. 11).
- the obtained perichondrium was cut with scissors, a scalpel and the like, and shaken in 0.3 * 0.3 collagenase at 37 * C for 3 hours. Thereafter, the mixture was centrifuged (1500 rpm / 5 min twice), and the precipitate was collected. As a result, perichondrial cells could be separated.
- the perichondrial cells obtained above were plated at 37: in Dulbecco's Modified Eagles' Medium / Nutrient Mixture F12 (DMEM / F12) and 103 ⁇ 4Fetal Bovine Serum (FBS), and the medium was changed twice a week. Within 2 weeks the cells grew and became confluent and were used for subculture. In 7-10 days the cells grew and became dense. The subculture could be continued for at least 6 months.
- DMEM / F12 Dulbecco's Modified Eagles' Medium / Nutrient Mixture F12
- FBS 103 ⁇ 4Fetal Bovine Serum
- Perichondrial cells were precipitated by centrifugation (twice at 1500 rpm / 5 min) to form a cell mass.
- Insulinlike growth factor-1 IGF-1
- bFGF Fibroblast growth factor
- 40ng / ml Dexamethasone 30-60 / xg / ml les ascorbic acid, l3 ⁇ 4Antibiotic ant imicot ic solution, l3 ⁇ 4Insul in-TransferrinSerine (ITS)
- IGF-1 Insulinlike growth factor-1
- bFGF Fibroblast growth factor
- Dexamethasone 30-60 / xg / ml les ascorbic acid
- l3 ⁇ 4Antibiotic ant imicot ic solution l3 ⁇ 4Insul in-TransferrinSerine (ITS)
- ITS l3 ⁇ 4Insul in-TransferrinSerine
- Chondrocytes were collected using blunt instruments such as exfoliants. Next, the cells were precipitated by centrifugation (twice at 1500 rpm / 5 min) to form cell clusters. The cell mass is 5 ng / ml Insul inl ike growth factor-1 (IGF-1), 5 ng / ml basic Fibroblast growth factor (bFGF), 40 ng / ml Dexamethasone, 30-60 g / ml L-ascorbic acid, The cells were cultured in serum-free medium of DMEM / F1.2 containing Antibiotic antiniicotic solution, Insul in ⁇ Transferrin ⁇ Serine (ITS) for 3-4 weeks.
- IGF-1 Insul inl ike growth factor-1
- bFGF basic Fibroblast growth factor
- Dexamethasone 40 ng / ml Dexamethasone
- perichondrial cells were seeded on the plated perichondrocytes in a dense state.
- Medium is 103 ⁇ 4 in DMEM / F12; FBS and lS! Antibiotic ic antimicotic solut ion or 10% FBS in DMEM / F12, l3 ⁇ 4Antibiotic ant imicotic solut ion, 5ng / ml IGF-l, 5ng / ml bFGF containing 40 ng / ml Dexamethasone was used.
- RT-PCR primers are type I collagen F: atgctcagct ttgtggatacgcgg (layout 1 J number 1), R: aggaaagccacgagcaccctgtgg (sequence number 2), type II collagen F: catcattgacattgcacccatg (sequence number 3), R: t tagt t tcctgtctctgctg (Rice 'J number 4), Acrican F: caggtgaagactttgtggacatcc (SEQ ID NO: 5), R: cctcctcaaaggtcagcgagtagc (SEQ ID NO: 6) was used.
- Quantitative PCR flyers are Taqman Gene Expression Assays (Applied Biosystems) type I collagen: Hs00266273—ml, type II collagen:
- type I collagen which is a perichondrial marker
- type I collagen a cartilage marker
- ELISA Enzyme Linked Immunosorbent Assay
- the obtained perichondrial cells and chondrocytes were subjected to a colony assembly. Each cell was seeded on a 35-screen easy grip cell culture dish adjusted to kells / cm 2 . Cells were set to Dulbecco's modified Eagle medium and Ham's F-12 medium containing 10% fetal bovine serum, 1% Antibiotic Antimycotic Solution, gas phase conditions were 37, and C0 2 concentration was 5 3 ⁇ 4; Culture was performed in an incubator. The medium was changed once every 3 days from 24 hours after sowing. After culturing for one month, the formed colonies were confirmed under a microscope and photographed. As a result, the perichondrial cell colonies were larger than the chondrocyte colonies (Fig. 12).
- perichondrium cells perichondrium-cartilage transition part cells, and chondrocytes were subjected to a colony assay. Each cell was seeded in a 35 mm easy grip cell culture dish adjusted to 52 cells / cm2. Cells are 10% fetal bovine serum, 1% Antibiotic Antimycotic Solution Dulbeccos modified agle medium and Ham s
- F- using 12 medium were cultured in a vapor phase condition 37, C0 2 incubator in the evening, which was set to a concentration of 5%.
- the medium was changed once every 3 days from 24 hours after sowing. After culturing for 14 days, the number of colonies was counted. More than 50 cell populations were taken as one colony. When the number of colonies was compared, peritoneal cells, perichondrium-cartilage transition zone cells, and chondrocytes showed higher colony-forming ability (Fig. 13).
- the long-term proliferative ability of the perichondrium cells, perichondrium-cartilage transition zone cells, and chondrocytes was examined. Each cell was seeded in a 35 mm easy grip cell culture dish at 1200 cells / cm2. Cells 10% fetal bovine serum, 1% Antibiotic Antimycotic Solution using Dulbecco's mo dined Eagle medium and Ham s F- 12 medium which contains 3 ⁇ 4 a, 37 a gas phase condition, C0 2 Incubate set at 5% concentration one Culture was performed in the evening. The medium was changed once every 3 days from 24 hours after sowing.
- Induction of differentiation into chondrocytes was performed by stratification culture of perichondrial cells and chondrocytes. Each cell was adjusted to 2.5 ⁇ 10 4 cells / cm 2 and seeded. After culturing for 7 days, the cells were detached using Hank's balanced solution containing 0.2 3 ⁇ 4 collagenase. Type II. Then, the cells were adjusted to 2.5 ⁇ 10 4 cells / cm 2 and overlaid on the cells cultured for 7 days and seeded. Up to 48 hours after sowing, culture with Dulbeccos modified Eagle medium and Ham s F-12 medium containing 10% fetal bovine serun 1% Antibiotic Antimycotic Solution.
- the perichondrial cell-derived chondrocytes derived from chondrocytes induced to differentiate into cartilage using a differentiation-inducing medium were detached with a cell lifter.
- the detached cells were collected in a 2.5 ml syringe (Terumo, Japan) equipped with a 23 G injection needle (Terumo, Japan).
- Severe immunodeficient mice (Sankyo, Japan) that had undergone depilation were injected subcutaneously with 1 ml of cells in the back of the mice, and transplanted. After transplantation, they were removed at 1 and 3 months and examined histologically. Hematosylin-Ejin, Alcian Blue 1, Elastica Wangyson, type I and type II collagen were stained, respectively.
- the perichondrial cells and the chondrocyte-derived tissue were both stained with Alcian blue and Elastica one-gisson (B, C, F, and G in FIGS. 17A and 17B). Furthermore, the tissue derived from perichondrial cells was stained with type I collagen and type II collagen (D in both FIGS. 17A and 17B). On the other hand, the chondrocyte-derived tissue was stained with type II collagen but not with type I collagen (Fig. 17A, H for both B).
- the number of cells per mm 2 in the cartilage part of the tissue removed at 1 month and 3 months after transplantation obtained by histological examination of cartilage tissue regenerated from human perichondrium and chondrocytes in vivo as described above. Measured. In the tissue derived from perichondrial cells, the number of cells did not change at 1 month and 3 months after transplantation, whereas in the tissue derived from chondrocytes, the cell number decreased at 3 months after transplantation. (Fig. 18).
- the obtained perichondrium cells, perichondrium-cartilage transition part cells, and chondrocytes were subjected to a colony assay. Each cell was seeded after adjusting to 500 cells per 35 mm easy grip cell culture dish.
- the cells were 10% human autologous serum, 1% Antibiotic Antimycotic Solution 3 ⁇ 4: containing 3 ⁇ 4 “5 Dulbeccos modined Eagle medium and Hams F-12 medium, and the gas phase conditions were set to 37: C0 2 concentration 5% Cultivation was performed in the incubator 24 hours after seeding and once every 7 days after seeding 9 days after culturing, photographs of colonies were taken ( Figure 19). Compared to chondrocytes, large colonies formed on the 9th day of culture.
- each cell was adjusted to 5000 cells per well of a 24-well cell culture plate and seeded.
- the cells used Dulbecco's modified Eagle medium and Ham's F-12 medium containing 10% baboon autologous blood iff, 1% Antibiotic Antimycotic Solution, in an incubator with a gas phase of 37 and a C0 2 concentration of 5%
- the culture medium was changed once every 3 days from 24 hours after seeding, and after 10 days of culture, photographs were taken (Fig. 20). Compared to% urine serum medium, the cells became confluent more quickly.
- human chondrocytes are proliferated and differentiated, and human chondrocytes are cultured. It became possible. Furthermore, human chondrocyte mass can be obtained by centrifuging tube culture, three-dimensional culture of human perichondrial cells with collagen gel, etc., or stratified culture.
- the human chondrocyte mass obtained as it is or embedded in a cartilage treatment material such as collagen gel and transplanted can be used for diseases related to cartilage (for example, congenital auricular malformations, costal cartilage defects, deformable knee joints). Can be used for articular cartilage damage (eg, knee osteoarthritis), tracheal cartilage defect, etc.
- treatments for aesthetic improvement in the cosmetic surgery field such as rhinoplasty, genioplasty, facial indentation, facial asymmetric correction, eyelid correction and facial cosmetic cartilage It is also useful for the treatment of transplantation.
- SEQ ID NO: 1 shows the base sequence of RT-PCR forward primer of type I collagen. Sequence number 2>
- SEQ ID NO: 2 shows the nucleotide sequence of RT-PCR reverse primer for type I collagen. ⁇ SEQ ID NO: 3>
- SEQ ID NO: 3 shows the nucleotide sequence of an RT-PCR forward primer for type I collagen.
- SEQ ID NO: 4 shows the base sequence of RT-PCR reverse primer for type I collagen.
- SEQ ID NO: 5 shows the nucleotide sequence of RT-PCR forward primer for aggrecan. SB column number 6>
- SEQ ID NO: 6 shows the nucleotide sequence of RT-PCR reverse primer for aggrecan.
Abstract
Description
Claims
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US12/524,359 US9951312B2 (en) | 2007-01-23 | 2008-01-23 | Method for preparation of cartilage cell |
EP08704109.1A EP2119767B1 (en) | 2007-01-23 | 2008-01-23 | Method for preparation of cartilage cell |
CN200880002777.4A CN101657536B (zh) | 2007-01-23 | 2008-01-23 | 软骨细胞制备方法 |
JP2008555127A JP4748222B2 (ja) | 2007-01-23 | 2008-01-23 | 軟骨細胞調製方法 |
KR1020097015300A KR101503939B1 (ko) | 2007-01-23 | 2008-01-23 | 연골 세포 조제 방법 |
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JP2013202014A (ja) * | 2012-03-29 | 2013-10-07 | Jms Co Ltd | 間葉系幹細胞の分化促進剤およびそれを用いた分化促進方法 |
WO2014148592A1 (ja) * | 2013-03-21 | 2014-09-25 | 公立大学法人横浜市立大学 | 軟骨細胞の調製方法 |
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EP2468827B1 (en) | 2010-12-21 | 2014-03-12 | Agfa-Gevaert | A dispersion comprising metallic, metal oxide or metal precursor nanoparticles |
CN104511052A (zh) * | 2014-12-16 | 2015-04-15 | 温州医科大学附属第一医院 | 一种骨膜生物支架与异体种子细胞复合的培养方法 |
EP3417888A1 (de) * | 2017-06-25 | 2018-12-26 | co.don AG | Verfahren zum herstellen von transplantierbarem knorpelgewebe |
WO2023032441A1 (ja) * | 2021-08-31 | 2023-03-09 | 公立大学法人横浜市立大学 | 造形可能かつ足場不要な軟骨組織の創出法 |
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Cited By (4)
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JP2013202014A (ja) * | 2012-03-29 | 2013-10-07 | Jms Co Ltd | 間葉系幹細胞の分化促進剤およびそれを用いた分化促進方法 |
WO2014148592A1 (ja) * | 2013-03-21 | 2014-09-25 | 公立大学法人横浜市立大学 | 軟骨細胞の調製方法 |
JPWO2014148592A1 (ja) * | 2013-03-21 | 2017-02-16 | 公立大学法人横浜市立大学 | 軟骨細胞の調製方法 |
US10100274B2 (en) | 2013-03-21 | 2018-10-16 | Public University Corporation Yokohama City University | Method for preparing chondrocytes |
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JPWO2008091013A1 (ja) | 2010-05-20 |
CN101657536B (zh) | 2016-06-01 |
JP4748222B2 (ja) | 2011-08-17 |
US20090324560A1 (en) | 2009-12-31 |
CN101657536A (zh) | 2010-02-24 |
US9951312B2 (en) | 2018-04-24 |
EP2119767B1 (en) | 2016-01-20 |
EP2119767A1 (en) | 2009-11-18 |
EP2119767A4 (en) | 2011-01-12 |
KR20090104833A (ko) | 2009-10-06 |
KR101503939B1 (ko) | 2015-03-18 |
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