JP-7856976-B2 - Cell cultures, methods for evaluating cell cultures, methods for producing cell cultures, and markers for evaluating cartilage-like tissue formation characteristics.

Inventors

• 佐藤 正人
• 高橋 匠
• 川口 祐加
• 佐藤 千香子
• 角田 智志
• 遠山 千春
• 宋 丹丹
• 宮沢 道秀

Assignees

• 学校法人東海大学
• 株式会社セルシード

Dates

Publication Date

20260512

Application Date

20210312

Priority Date

20200313

Claims (1)

1. A method for evaluating cell cultures, Regarding the cell population contained in the cell culture, The method includes a determination step of determining whether the expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b is below a threshold for each surface marker, and/or whether the expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD120a, CD201, EGFR, CD146, and CD140a is above a threshold for each surface marker. The process further includes an analysis step of analyzing the aforementioned cell population by flow cytometry, The determination in the determination step is made based on the analysis results in the analysis step. The expression intensity is the standardized mean fluorescence intensity measured by analyzing the cell population by flow cytometry, and the standardized mean fluorescence intensity satisfies at least one of the following conditions 1 to 10 and/or satisfies at least one of the following conditions 11 to 16. In the determination step, A sheet-like cell culture is determined to have cartilage-like tissue formation characteristics if the expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b is below the threshold for each surface marker, and/or the expression intensity of at least one cell surface marker selected from the group consisting of CD26 , CD120a, CD201, EGFR, CD146, and CD140a is above the threshold for each cell surface marker. The aforementioned evaluation method. Condition 1: When the cell population is labeled with an anti-CD166 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 240 or less. Condition 2: When the cell population is labeled with an anti-CD165 antibody conjugated with BB700, the standardized mean fluorescence intensity derived from BB700 is 132 or less. Condition 3: When the cell population is labeled with an anti-CD99 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.9 or less. Condition 4: When the cell population is labeled with an anti-GD2 antibody conjugated with BB700, the standardized mean fluorescence intensity derived from BB700 is 3.6 or less. Condition 5: When the cell population is labeled with an anti-STRO-1 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.4 or less. Condition 6: When the cell population is labeled with an anti-CD108 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 1.9 or less. Condition 7: When the cell population is labeled with an anti-CD164 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 3.0 or less. Condition 8: When the cell population is labeled with an anti-CD6 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.4 or less. Condition 9: When the cell population is labeled with an anti-CD106 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 2.4 or less. Condition 10: When the cell population is labeled with an anti-CD107b antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 2.0 or less. Condition 11: When the cell population is labeled with an anti-CD26 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 2.3 or higher. Condition 12: When the cell population is labeled with an anti-CD120a antibody conjugated with APC, the standardized mean fluorescence intensity derived from the APC is 24 or higher. Condition 13: When the cell population is labeled with an anti-CD201 antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 18 or higher. Condition 14: When the cell population is labeled with an anti-EGFR antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 3.2 or higher. Condition 15: When the cell population is labeled with an anti-CD146 antibody conjugated with FITC, the standardized mean fluorescence intensity derived from the FITC is 1.6 or higher. Condition 16: When the cell population is labeled with an anti-CD140a antibody conjugated with PE, the standardized mean fluorescence intensity derived from the PE is 5.6 or higher.

Description

This technology relates to cell cultures, methods for evaluating cell cultures, methods for producing cell cultures, and markers for evaluating cartilage-like tissue formation characteristics. More specifically, this technology relates to cell cultures containing a cell population expressing a specific cell surface marker, methods for evaluating cell cultures based on a specific cell surface marker, methods for producing cell cultures containing a cell population expressing a specific cell surface marker, and specific cell surface markers used for evaluating cartilage-like tissue formation characteristics. Regarding the treatment of musculoskeletal disorders such as osteoarthritis, cartilage tissue therapy using tissue regeneration engineering technology is being employed. In this treatment, cultured chondrocytes or cartilage tissue created from chondrocytes can be transplanted to the affected area. Various transplant materials have been proposed to date. For example, Patent Document 1 below describes "a transplant material for transplantation to a predetermined transplantation site, wherein cells corresponding to the predetermined transplantation site are held in a cell-holding carrier obtained by subjecting a tissue structure of the same type as the predetermined transplantation site, obtained from somatic tissue, to an antigenicity suppression treatment while maintaining the shape of the tissue structure." (Claim 1) Japanese Patent Publication No. 2003-180819 This is a flowchart of the experiment.This graph shows the gene expression ratios of COL2A1 and COL1A1.This figure shows the staining results. 1. cell culture The present invention provides a cell culture having cartilage-like tissue formation characteristics, and more particularly a cell culture having hyaline cartilage-like tissue formation characteristics. The cell population contained in the cell culture has an expression intensity of at least one cell surface marker selected from the group consisting of CD166, CD165, CD99, GD2, STRO-1, CD108, CD164, CD6, CD106, and CD107b that is below a threshold for each cell surface marker, and/or the expression intensity of at least one cell surface marker selected from the group consisting of CD26, CD73, CD105, CD44, CD120a, CD201, EGFR, CD146, CD140a, and CD90 that is above a threshold for each cell surface marker. By having these cell surface markers above or below a predetermined threshold, the cell culture is suitable for cartilage repair, and more particularly suitable for knee cartilage repair. The cell culture of the present invention has the property of forming cartilage-like tissue, and more particularly, hyaline cartilage-like tissue. The cell culture of the present invention has the property of forming cartilage-like tissue, and more particularly, hyaline cartilage-like tissue, when transplanted into a human living organism, for example, when transplanted into the articular cartilage portion of the knee of a human living organism. In this specification, "cartilage-like tissue" may be tissue having components and/or functions equivalent to or similar to cartilage tissue (particularly knee articular cartilage tissue), and for example, tissue that expresses type II collagen in the same way as hyaline cartilage tissue. In the present invention, preferably, the expression intensity of at least CD99 and/or GD2 among the cell surface markers is below a threshold for each of these cell surface markers. By having the expression intensity of CD99 and/or GD2 below a predetermined threshold, the cell culture can exhibit particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics. Furthermore, in the present invention, preferably, the expression intensity of at least CD26 and/or CD73 among the cell surface markers is above a threshold for each of these cell surface markers. By having the expression intensity of CD26 and/or CD73 above a predetermined threshold, the cell culture can exhibit particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics. Particularly preferable is that, among the cell surface markers, the expression intensity of at least CD99 and/or GD2 is below the threshold for each of these cell surface markers, and the expression intensity of at least CD26 and/or CD73 is above the threshold for each of these cell surface markers. When these four cell surfaces satisfy the criteria for the thresholds, the cell culture can exhibit particularly good cartilage-like tissue formation characteristics, especially hyaline cartilage-like tissue formation characteristics. Furthermore, in the present invention, preferably, the expression intensity of at least one, two, or three of the cell surface markers, CD26, CD73, and CD44, is above a certain threshold. By having the expression intensity of one, two, or three of the CD26, CD73, and CD44 above a predetermine