Method for production of t cell population
A cell group and cell technology, applied in the field of preparing T cell groups useful in the medical field, can solve problems such as inability to obtain sufficient effects
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Embodiment 1
[0138] Example 1CD45RA + CD62L + Analysis of T cells
[0139] (1) Isolation and preservation of PBMC
[0140] Blood components were collected from healthy human donors who gave informed consent, and then the collected blood was diluted 2-fold with phosphate-buffered saline (hereinafter referred to as PBS), spread on Ficoll-paque (manufactured by Amasham Biosystems), and fed at 600 × g Centrifuge for 20 minutes. Peripheral blood mononuclear cells (hereinafter referred to as PBMCs) in the middle layer were collected by a pipette and washed. The collected PBMCs were suspended in a preservation solution containing 90% FBS (manufactured by Cambrex) / 10% DMSO, or CP-1 (manufactured by Jidong Pharmaceutical Co., Ltd.) containing 8% human serum albumin (manufactured by Baxter, hereinafter referred to as HSA). ) and RPMI1640 medium (manufactured by Sigma) in an equal volume mixture, and stored in liquid nitrogen. When expanding T cell culture, these preserved PBMCs were rapidly tha...
Embodiment 2
[0155] Example 2CD45RA + CCR7 + Analysis of T cells
[0156] (1) Expanded culture of T cell population
[0157] The PBMC prepared in Example 1-(1) was suspended in AIM-V (hereinafter referred to as 3% AIM-V) containing 3% human AB serum, and the concentration was 1×10 6 cells / mL, prepare the cell solution, and then add 3% AIM-V to the plate prepared in Example 1-(2) immobilized with anti-human CD3 antibody, or immobilized with anti-human CD3 antibody and CH To the -296 plate, add the above-mentioned cell solution at 1 mL / well, respectively. Add IL-2 to a final concentration of 1000 U / mL and place the plates in 5% CO 2 , and cultured at 37°C (cultivation day 0). On the 4th day from the beginning of culture, each group was diluted with AIM-V containing 1% human AB serum (volume 6mL), the concentration was 0.075×10 6 cells / mL, transfer the culture medium to a 12.5cm cell without any fixation 2 In the cell culture flask, add IL-2 with a final concentration of 500U / mL. The ...
Embodiment 3
[0175] Example 3 Using CH-296 to expand CD45RA in the cultured T cell population + CCR7 + T cells and CD45RA - CCR7 - Analysis of cytokine production of T cells
[0176] (1) Expanded culture of T cell population
[0177] Suspend the PBMC prepared in Example 1-(1) in GT-T503 medium (manufactured by TAKARA BIO, hereinafter referred to as 0.5% GT-T503) containing 0.5% human AB serum and 0.2% HSA, at a concentration of 0.25 ×10 6 cells / mL, prepare the cell solution, then add 0.5% GT-T503 to the plate immobilized with anti-human CD3 antibody prepared in Example 1-(2), or immobilized with anti-human CD3 antibody and To the CH-296 plate, the above-mentioned cell solution was added at 1 mL / well. Add IL-2 to a final concentration of 1000 U / mL and place the plates in 5% CO2 , and cultured at 37°C (cultivation day 0). On the 4th day after the start of culture, the culture solution of each group was diluted about 8 times with 0.5% GT-T503, and 6 mL of the diluted solution was trans...
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