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Method for producing lymphocyte cell group consisting mainly of memory t cells

A technology of lymphocytes and manufacturing methods, applied in the direction of animal cells, vertebrate cells, blood/immune system cells, etc.

Active Publication Date: 2014-12-10
LYMPHOTEC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the current situation, there is no method for easily collecting a large amount of memory T cells required for clinical supply without imposing a large burden on the lymphocyte provider.

Method used

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  • Method for producing lymphocyte cell group consisting mainly of memory t cells
  • Method for producing lymphocyte cell group consisting mainly of memory t cells
  • Method for producing lymphocyte cell group consisting mainly of memory t cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] [Activation and proliferation of lymphocytes]

[0072] (1) Preparation of peripheral blood lymphocytes

[0073]50 mL of peripheral blood was collected intravenously from 3 healthy people who had obtained consent by adding heparin. Each blood test sample was transferred to a 250 mL centrifugal sedimentation tube (BD Falcon; 352075), and 100 mL of washing solution (physiological saline supplemented with 0.1% human albumin: 500 mL of normal saline, manufacturer : Otsuka Pharmaceutical, 2.0mL 25% human blood albumin, manufacturer: Tanabe Mitsubishi Pharmaceutical), slowly mixed, diluted 3 times. The following operations including this one are performed under sterile conditions. For each test sample, 3-fold diluted blood was relayered in four 50 mL centrifugal sedimentation tubes (BD Falcon; 352070) filled with 15 mL of polysucrose (Ficoll) (manufactured by GE Healthcare Bioscience). The centrifugal precipitation tube was continuously centrifuged at 1800 rpm for 20 minute...

Embodiment 2

[0103] [Changes in Lymphocyte Surface Antigens During Activation Culture]

[0104] (1) Preparation and activation culture of lymphocytes

[0105] By the same procedure as in (1) of Example 1, peripheral blood lymphocytes were prepared from 3 healthy individuals who had obtained consent, and the number of cells was counted.

[0106] In the same manner as in (3) of Example 1, the prepared lymphocytes were centrifuged, and the cell density of the lymphocytes was adjusted to 6×10 with medium 1. 5 individual / mL. 50mL of this cell suspension was injected into the 225cm cell suspension made in (2) of Example 1. 2 In the OKT3 immobilized flask (manufactured by Sumitomo Bakelite Co., Ltd.: MS-2180R), medium 1 was used in the same manner as in Example 1 (3) in TE-HER type CO 2 In the incubator (manufactured by HIRASAWA Co., Ltd.), the temperature is 37.0±0.5°C, the humidity is 95.0±5.0%, CO 2 The concentration was 5.0±0.2% for cultivation. During the culture, suspended cells were c...

Embodiment 3

[0119] [Research on the density of frozen cells in the freezing process]

[0120] The preparation containing the T cell population obtained by the method of the present invention will be used for treatment, and the number of finally obtained lymphocytes is high to be effective. Therefore, the possibility that cryopreservation affects the number of cells per test tube after activation expansion culture was investigated.

[0121] (1) Study on frozen cell density-1 activation culture

[0122] By the same procedure as in (1) of Example 1, peripheral blood lymphocytes were prepared from 3 healthy individuals who had obtained consent, and the number of cells was counted.

[0123] In the same manner as in (3) of Example 1, the prepared lymphocytes were centrifuged, and the cell density of the lymphocytes was adjusted to 6×10 with medium 1. 5 individual / mL. 50mL of this cell suspension was injected into the 225cm cell suspension made in (2) of Example 1. 2 In the OKT3 solid-phase ...

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Abstract

Approximately 50% of lymphocytic cells derived from peripheral blood is occupied by naive T cells before an activation culture is performed, but the ratio decreases through the activation culture. The occupancy of the memory T cells, which is less than half before the start of the culture, is increased by an activation culture to reach 95%, but decreases if the culture continues. An object of the present invention is to prepare CD45RO positive memory T cell groups in quantity without having to use an antibody or the like, conveniently, and rapidly, and to provide a pharmaceutical composition consisting mainly of the memory T cell groups prepared by the method. According to the present invention, harvested lymphocytic cells are activation-cultured, and the occupancy of the memory T cells in the lymphocyte cell group reaching a desired value is detected through the index of the density of the cultured lymphocyte cell group exceeding a predetermined threshold, the related cell group is harvested for free preservation, and the related free-preserved cell is thawed, cultured, and grown so as to produce the lymphocyte cell groups consisting mainly of the memory T cells in quantity.

Description

technical field [0001] The present invention relates to a method for mass production of a lymphocyte population mainly composed of memory T cells, a pharmaceutical composition containing the lymphocyte population, and a production system (combination of means such as a device) for the lymphocyte population, etc. The mass production method is a method for easily obtaining a large amount of a CD45RO-positive and CD62L-positive T cell population and a CD45RO-positive and CD62L-negative T cell population from peripheral blood containing T cells. Background technique [0002] Lymphocytes have attracted much attention in recent years as an important means of supporting the immune system for the body's defense. Lymphocytes express antigenic types called differentiation antigens on their surfaces, and lymphocytes are classified into various subsets according to the antigenic types. For example, the presence or absence of CD3 (CD is an abbreviation for cluster of differentiation) ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61P31/00A61P35/00A61P37/00C12N5/0786A61K35/14A61K35/17A61P31/12A61P31/18
CPCA61K35/17C12N5/0636A61P31/00A61P31/12A61P31/18A61P35/00A61P37/00A61K39/4611A61K39/464499
Inventor 关根晖彬大隅一兴小森启一郎清水则夫
Owner LYMPHOTEC
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