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WO2016013352A1 - Procédé de production d'une cellule adipeuse dédifférenciée - Google Patents

Procédé de production d'une cellule adipeuse dédifférenciée Download PDF

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Publication number
WO2016013352A1
WO2016013352A1 PCT/JP2015/068658 JP2015068658W WO2016013352A1 WO 2016013352 A1 WO2016013352 A1 WO 2016013352A1 JP 2015068658 W JP2015068658 W JP 2015068658W WO 2016013352 A1 WO2016013352 A1 WO 2016013352A1
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Prior art keywords
adipocytes
dedifferentiated
cells
less
mature
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PCT/JP2015/068658
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English (en)
Japanese (ja)
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雅規 本田
仁奈 鶴町
大輔 秋田
守雄 外木
浩一郎 加野
太郎 松本
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学校法人日本大学
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Priority to JP2016520701A priority Critical patent/JP5991687B2/ja
Publication of WO2016013352A1 publication Critical patent/WO2016013352A1/fr

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  • the present invention relates to a method for producing dedifferentiated fat cells (DFAT cells). More specifically, the present invention relates to a method for isolating mature adipocytes by treating adipose tissue with a specific concentration of collagenase and producing dedifferentiated adipocytes from the mature adipocytes.
  • DFAT cells dedifferentiated fat cells
  • the present inventors have isolated mature adipocytes from adipose tissue and produced dedifferentiated adipocytes from the mature adipocytes.
  • Dedifferentiated adipocytes are cells that have re-acquired pluripotency obtained by spontaneously initiating dedifferentiation of mature adipocytes by culturing isolated mature adipocytes by a ceiling culture method. It is considered useful as a cell source for regenerative medicine.
  • it is important to efficiently isolate mature adipocytes from adipose tissue under conditions that do not damage the cells as much as possible. Therefore, the present inventors examined the optimum collagenase concentration in the treatment of adipose tissue for producing a large amount of dedifferentiated adipocytes in the present invention.
  • An object is to provide an efficient method for producing dedifferentiated adipocytes.
  • the inventors of the present invention have found that the concentration of collagenase conventionally used at a concentration of 0.1 w / v% in the isolation of mature adipocytes from adipose tissue Matured adipocytes can be collected at a efficiency about 3 times higher than the conventional concentration when the concentration is higher than 0.01 w / v% and lower than 0.1 w / v%. Has been found to be able to be produced, and the present invention has been completed.
  • the present invention relates to the following (1) to (6) methods for producing dedifferentiated adipocytes.
  • a method for producing dedifferentiated adipocytes comprising the following step A.
  • A. A step of isolating mature adipocytes by treating adipose tissue with collagenase at a concentration higher than 0.01 w / v% and lower than 0.1 w / v% (2) The concentration of collagenase is 0.02 w / v
  • the production method according to (1) wherein the production method is v% or more and 0.05% or less.
  • the present invention it becomes possible to collect mature adipocytes with high efficiency without damaging isolated mature adipocytes.
  • the cost can be reduced by reducing the amount of collagenase used in the treatment.
  • the “method for producing dedifferentiated adipocytes” of the present invention may be any method for producing dedifferentiated adipocytes, including the following step A, and includes other steps useful for efficient production of dedifferentiated adipocytes. It may be included.
  • adipose tissue obtained from subcutaneous or internal organs is treated with collagenase at a concentration higher than 0.01 w / v% and lower than 0.1 w / v%.
  • the enzyme solution containing mature adipocytes obtained by this collagenase treatment is filtered with a mesh having a pore size of 100 and 150 ⁇ m, and a single fraction consisting of only monocystic adipocytes which are mature adipocytes For example, isolation of mature adipocytes.
  • the concentration of collagenase may be higher than 0.01 w / v% and lower than 0.1 w / v%, particularly 0.02 w / v% or more and 0.05% or less. Is preferred.
  • the mature adipocytes to be produced may have a normal size of about 60 to 100 ⁇ m, but are preferably less than 60 ⁇ m, particularly preferably less than 40 ⁇ m.
  • the “dedifferentiated adipocyte production method” of the present invention may further include the following step B.
  • the isolated mature adipocytes are cultured by a ceiling culture method. Can be mentioned.
  • the present invention of the present inventors Patent Nos. 5055611 and 5055613 may be referred to.
  • the “method for producing mesenchymal cells” of the present invention may be any method for producing mesenchymal cells, including the step of inducing differentiation of dedifferentiated adipocytes obtained by “method for producing dedifferentiated adipocytes”. Other processes useful in the production of mesenchymal cells may be included. “Mesenchymal cells” include osteoblasts, myoblasts, cardiomyocytes, endothelial cells, chondrocytes or adipocytes. In the step of inducing differentiation of dedifferentiated adipocytes, any conventionally known methods such as agents and methods useful for inducing differentiation can be used in accordance with the cells to be differentiated.
  • Step A Step of isolating mature adipocytes from adipose tissue 5 g of adipose tissue collected from the buccal fat pad located under the human buccal mucosa is added with collagenase (Type II; SIGMA) and NaHCO 3 -containing Dulbecco's modified Eagle medium (DMEM; SIGMA) And treated with collagenase.
  • collagenase Type II; SIGMA
  • DMEM Dulbecco's modified Eagle medium
  • the collagenase concentration in the medium was 0.02 w / v%, and a comparison was made with a concentration of 0.01 w / v%, 0.10 w / v%, or 0.50 w / v% for comparison.
  • the solution was filtered through a nylon mesh to obtain a cell suspension.
  • the obtained cell suspension is centrifuged at 700 G for 1 minute, the monocystic fat fraction separated in the upper layer is added to fresh DMEM medium supplemented with 10% FBS, and the centrifugation at 700 G is repeated 3 times for 1 minute.
  • mature adipocytes were obtained as monocystic adipocytes.
  • Step B Step of obtaining dedifferentiated adipocytes from mature adipocytes
  • the monocystic adipocytes obtained by step A are transferred to a tissue culture flask (Falcon, 3107), and are added to a DMEM medium supplemented with 20% FBS, 1% penicillin / streptomycin.
  • the flask was completely filled, and it was left to stand in a carbon dioxide culture apparatus adjusted to a gas phase of 37 ° C., 5% CO 2 , 95% air, and cultured for 7 days. After 4 days of culture, most of the cells adhered firmly to the ceiling surface of the flask, and the morphology changed to multivesicular adipocytes having lipid droplets of various sizes around the large lipid droplets.
  • the lipid droplets became even smaller, and many cells that changed to a fibroblast-like morphology without any lipid droplets were observed.
  • the medium in the flask was replaced with 20% FBS-added DMEM medium, and culturing was continued for 10 days in a carbon dioxide culture apparatus so that the cell adhesion surface was the bottom. The medium was changed every 4 days. Cells without lipid droplets proliferated actively, and after 10 days in culture, the cells in the flask became only fibroblast-like cells and reached confluence.
  • This fibroblast-like cell has an active proliferative ability and also has a differentiation ability to redifferentiate into adipocytes having lipid droplets by a differentiation inducer such as DEX, INS, IBMX, etc. It was created as a cell-derived dedifferentiated adipocyte.
  • the produced dedifferentiated adipocytes were resuspended in DMEM medium supplemented with 20% FBS so as to be 1 ⁇ 10 4 cells / ml. Thereafter, the cells were seeded in a culture flask for tissue culture (Falcon, 3001) coated with collagen type II, and left in a carbon dioxide culture apparatus adjusted to a gas phase of 37 ° C., 5% CO 2 and 95% air. And cultured. The medium was changed every 4 days. After 8 days of culture, the number of cells was counted when the dedifferentiated adipocytes of the present invention reached confluence (FIGS. 1-3, P1). Thereafter, the second subculture was performed and the number of cells was similarly counted (FIGS. 1 to 3, P2), and then the third subculture was performed and the number of cells was similarly counted (FIGS. 1 to 3). , P3).
  • Example 2 Method for producing dedifferentiated adipocytes (2) 1. Comparison of the number of mature adipocytes A collagen tissue treatment was performed on adipose tissue collected from human buccal fat pad in the same manner as in Example 1. After treatment with collagenase at each concentration for 1 hour, extracellular matrix components were removed using a 100 ⁇ m cell strainer (BD FALCON), followed by filtration and low-speed centrifugation to obtain a mature adipocyte fraction. The number of cells contained in 200 ⁇ l of the floating cell fraction at each collagenase concentration was measured using a Coulter Counter (registered trademark) Multisizer 3 (Beckman Coulter, Miami, FL, USA), and the number of mature fat cells collected. Compared. This measurement was performed three times for each concentration.
  • BD FALCON 100 ⁇ m cell strainer
  • the number of cells contained in 200 ⁇ l of the floating cell fraction at each collagenase concentration was measured using a Coulter Counter (registered trademark) Multisizer 3 (Beckman Co
  • the number of mature adipocytes obtained by the above 1 and 2 is shown in FIG.
  • mature fat cells having a size in the range of 60 ⁇ m or more and less than 100 ⁇ m were collected in the same number of mature fat cells at any collagenase concentration.
  • mature adipocytes with a size of less than 60 ⁇ m can be collected in a significantly larger amount than other concentrations when the collagenase concentration is 0.02 w / v%.
  • mature adipocytes having a size of less than 40 ⁇ m can be collected 6 times or more when the collagenase concentration is 0.02 w / v% compared to other concentrations.
  • Fluorescent staining The fraction of cells less than 40 ⁇ m or the fraction of cells less than 40 ⁇ m and less than 100 ⁇ m collected in Adipore (Lonza, Walkerslive, MD, USA) positive for lipid droplets (triglycerides) and Hoechst 33342 (Sigma) positive for nuclei -Aldrich, 5 ⁇ g / ml) was stained for 20 minutes and observed with a fluorescence microscope (KEYENCE). As a result, as shown in FIG. 5, both the cell fraction of less than 40 ⁇ m (FIG. 5, A) and the cell fraction of 40 ⁇ m to less than 100 ⁇ m (FIG. 5, B) were stained with triglycerides and nuclei. It was confirmed to be a mature adipocyte.
  • the number of mature adipocytes of less than 40 ⁇ m is significantly larger than that of mature adipocytes of 40 ⁇ m or more and less than 100 ⁇ m from the 6th day of the ceiling culture, and dedifferentiated adipocytes are removed. It was confirmed that it differentiated.
  • CD13 PE-CD13
  • CD44 FITC-CD44
  • CD45 FITC-CD45
  • CD73 PE-CD73
  • CD90 APC-CD90
  • CD105 APC-CD105
  • the reaction was performed with CD146 (PE-CD146), CD271 (PE-CD271), and STRO-1 (FITC-STRO1) (all of which were BD Biosciences, San Jose, CA, USA) for 20 minutes. Thereafter, the cells after the reaction were washed with PBS, and the percentage of positive cells was measured with a flow cytometer.
  • dedifferentiated adipocytes obtained from a cell fraction of less than 40 ⁇ m are more mesenchymal stem cells than dedifferentiated adipocytes obtained from a cell fraction of 40 ⁇ m or more and less than 100 ⁇ m. It was confirmed that the ratio of CD146 positive cells as a marker was about 1.5 to 2 times higher.
  • Example 3 Method for producing mesenchymal cells Subcultured dedifferentiated adipocytes obtained from the cell fraction of less than 40 ⁇ m obtained in Example 2 and dedifferentiated adipocytes obtained from the cell fraction of 40 ⁇ m or more and less than 100 ⁇ m, Cells from the first passage were seeded at 1 ⁇ 10 4 in a 12-well plate, and when they became confluent, induction culture was performed in an osteoblast induction medium for 21 days.
  • the composition of the differentiation-inducing medium (DIFFERENTIATION-INDUCING CULTURE MEDIUM (may be referred to as IM or Osteogenic medium in the drawing)) is the growth medium (Growth medium (may be indicated as GM in the drawing)) [DMEM, 10% (v / v) fetal bovine serum (FBS; 13B103, Sigma-Aldrich), 1% antibiotics] to 100 nM dexamethasone (Sigma-Aldrich), 10 mM b-glycerphosphine (Sigma-Aldrich-Sigma-Aldrich-Sigma-Aldrich-Sigma-Aldrich-Sigma-Aldrich-Sigma-Aldrich-Sigma-Albich-Sigma-Aldrich-Sigma-Albich Aldrich) is added. This osteoblast induction was evaluated by alkaline phosphatase activity, alizarin red S staining, and calcium quantitative test
  • alkaline phosphatase activity The cells at 0, 3, 5, 7, 10, 14 days after the start of induction were collected, the protein amount was measured using Pierce BCA protein Assay Reagent Kit (Thermo Scientific, Rockford, IL, USA) and alkaline phosphatase Activity was measured. As a result, as shown in FIG. 7, dedifferentiated fat cells obtained from the cell fraction of less than 40 ⁇ m (FIG. 7, under 40 ⁇ m IM) were obtained from the cell fraction of 40 ⁇ m or more and less than 100 ⁇ m. Compared to (FIG. 7, 40-100 ⁇ m IM), alkaline phosphatase activity was significantly higher on days 3, 5, and 7 after the start of induction.
  • the present invention it becomes possible to efficiently collect mature adipocytes without damaging the isolated mature adipocytes.
  • a large amount of dedifferentiated adipocytes can be produced, so that they can be widely used as a cell source for regenerative medicine.

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Abstract

Le problème abordé par cette invention est de pourvoir à un procédé d'isolement d'une cellule adipeuse mature d'un tissu adipeux à une efficacité élevée, et à la production d'une cellule adipeuse dédifférenciée à partir de la cellule adipeuse mature. La solution selon l'invention porte sur un procédé de production d'une cellule adipeuse dédifférenciée, ledit procédé impliquant l'étape (A) suivante : traiter un tissu adipeux avec 0,02 % p/v d'une collagénase pour isoler une cellule adipeuse mature.
PCT/JP2015/068658 2014-07-23 2015-06-29 Procédé de production d'une cellule adipeuse dédifférenciée WO2016013352A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105749347A (zh) * 2016-02-24 2016-07-13 广州赛莱拉干细胞科技股份有限公司 一种美容填充物及其制备方法
JP2020066624A (ja) * 2018-10-18 2020-04-30 学校法人日本大学 壊死性腸炎治療用組成物
WO2023190736A1 (fr) * 2022-03-29 2023-10-05 学校法人日本大学 Procédé de production de cellules adipeuses dédifférenciées humaines et milieu de culture pour la production de cellules adipeuses dédifférenciées humaines à partir de cellules adipeuses matures humaines

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* Cited by examiner, † Cited by third party
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JP7020667B2 (ja) * 2017-12-11 2022-02-16 学校法人日本大学 哺乳動物由来の脱分化脂肪細胞から神経細胞を製造する方法及び哺乳動物由来の脱分化脂肪細胞から神経細胞への分化誘導用キット
JP6826744B1 (ja) 2020-07-17 2021-02-10 医療法人Ｙａｎａｇａ ＣＬｉｎｉｃ 成熟脂肪細胞含有組成物の製造方法
EP4431104A1 (fr) * 2021-11-11 2024-09-18 Keio University Traitement de l'arthrose du genou en utilisant une lignée de cellules souches mésenchymateuses dérivées de tissu adipeux

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105749347A (zh) * 2016-02-24 2016-07-13 广州赛莱拉干细胞科技股份有限公司 一种美容填充物及其制备方法
JP2020066624A (ja) * 2018-10-18 2020-04-30 学校法人日本大学 壊死性腸炎治療用組成物
JP7348612B2 (ja) 2018-10-18 2023-09-21 学校法人日本大学 壊死性腸炎治療用組成物
WO2023190736A1 (fr) * 2022-03-29 2023-10-05 学校法人日本大学 Procédé de production de cellules adipeuses dédifférenciées humaines et milieu de culture pour la production de cellules adipeuses dédifférenciées humaines à partir de cellules adipeuses matures humaines

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