Direct venous re-transfusion immune cell cryopreservation medium and application thereof

A technology of immune cells and cryopreservation solution, applied in the field of biomedicine, which can solve the problems of cumbersome cryopreservation process and subsequent application procedures, unclear components of cryopreservation solution, and unsuitability for large-scale production, so as to facilitate clinical application and ensure safety Effectiveness and effectiveness, good effect of cryopreservation

Active Publication Date: 2017-05-17

HARBIN MEDICAL UNIVERSITY

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AI-Extracted Technical Summary

Problems solved by technology

[0010] The technical problem to be solved by the present invention is to overcome the poor cryopreservation effect, unclear composition of cryopreservation solution, relatively cumbersome cryopreservation process and subsequent application p...

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Abstract

The invention discloses a direct venous re-transfusion immune cell cryopreservation medium and application thereof. The direct venous re-transfusion immune cell cryopreservation medium is prepared from dimethyl sulfoxide, human serum albumin, dextran, hydroxyethyl starch, heparin sodium and other clinical medicinal level raw materials by optimizing the proportion of the dose of each raw material. In addition, the operation steps are simplified and the cryopreservation effect is improved by optimizing the cryopreservation method. On one hand, the potential risk of introducing an animal source antigenic substance in the cell treatment process is avoided, and on the other hand, the cryopreservation effect of immune cells is better than that of a conventional cryopreservation medium, thereby guaranteeing the safety and validity in clinical use. Two kinds of components are used for preservation, so that the cryopreservation medium can be preserved stably for a long time. The method disclosed by the invention is simple in operation, provides stronger operability for implementation of large-scale cryopreservation, and simultaneously provides a fundamental technical guarantee for implementation of immune cell treatment.

Application Domain

Dead animal preservation

Technology Topic

Human serum albuminPotential risk +10

Examples

Experimental program(3)

Example Embodiment

[0033] Example 1: Preparation and application of immune cell cryopreservation solution for direct intravenous infusion

[0034] 1. Raw materials and specifications

[0035] Hydroxyethyl starch and sodium chloride injection: 500ml: Hydroxyethyl starch (200/0.5) 30g

[0036] Dextran 40 glucose injection: 500ml: 30g dextran 40 and 25g glucose

[0037] Heparin Sodium Injection: 2ml: 12,500 units

[0038] Human albumin injection: 50ml: 10g total protein

[0039] 2. Preparation and application of immune cell cryopreservation solution for direct intravenous infusion

[0040] 1) Prepare liquid A and liquid B separately

[0041] A solution (100ml): 8.3ml dimethyl sulfoxide + 45.8ml hydroxyethyl starch sodium chloride injection + 45.8ml dextran 40 glucose injection + 0.1ml heparin sodium injection.

[0042] The content of each component in A solution is: dimethyl sulfoxide 8.3v/v%, hydroxyethyl starch 2.75w/v%, dextran 2.75w/v%, glucose 2.29w/v%, heparin sodium 625U/ml, The rest are medical injections.

[0043] B solution (100ml): 20w/v% human albumin injection.

[0044] 2) Mix liquid A and liquid B in a volume ratio of 3:2, and place in a refrigerator at 4°C for later use.

[0045] 3) Take 50ml of peripheral blood from healthy volunteers, slowly add it to a centrifuge tube containing lymphocyte separation solution, centrifuge at 500g for 20 minutes, and suck albuginea cells. Wash twice with PBS and count.

[0046] 4) According to the counting results, resuspend the PBMC cells with the pre-chilled cryopreservation solution prepared in 2) so that the cell density is 1×10 7 /ml, transfer the cells to the freezer bag.

[0047] 5) Place the cryopreservation bag containing the cells in a refrigerator at 4°C for 15 minutes.

[0048] 6) Place the cryopreservation bag containing the cells in the refrigerator at -80°C overnight.

[0049] 6) Store the cryopreservation bag containing the cells in liquid nitrogen.

[0050] 7) After 1, 6, and 12 months, remove the cells from the liquid nitrogen tank, place them in a 37°C water bath to thaw, and take a small amount of cells to detect the cell viability by trypan blue staining.

[0051] At the same time, PBMC cells treated with a control cryopreservation solution (50% FBS+40% RPMI1640+10% DMSO) were used as a control, except for the cryopreservation solution, other steps were the same as above.

[0052] 3. Results

[0053] The comparison of the resuscitation rate of PBMC with different cryopreservation solutions is shown in Table 1:

[0054] Table 1 Comparison of PBMC resuscitation rates with different cryopreservation solutions

[0055] Freezing liquid of the present invention Control Cryopreservation Solution 1 month survival rate 98.5％ 87.1％ 6-month survival rate 96.2％ 85.4％ 12 months live rate 93.7％ 80.7％

Example Embodiment

[0056] Example 2: Preparation and application of immune cell cryopreservation solution for direct intravenous infusion

[0057] 1. Raw materials and specifications

[0058] Hydroxyethyl starch and sodium chloride injection: 500ml: Hydroxyethyl starch (200/0.5) 30g

[0059] Dextran 40 glucose injection: 500ml: 30g dextran 40 and 25g glucose

[0060] Heparin Sodium Injection: 2ml: 12,500 units

[0061] Human albumin injection: 50ml: 5g total protein

[0062] 2. Preparation and application of immune cell cryopreservation solution for direct intravenous infusion

[0063] 1) Prepare liquid A and liquid B separately

[0064] A solution (100ml): 10ml dimethyl sulfoxide + 70ml hydroxyethyl starch sodium chloride injection + 19.9ml dextran 40 glucose injection + 0.1ml heparin sodium injection

[0065] The content of each component in A solution is: dimethyl sulfoxide 10v/v%, hydroxyethyl starch 4.2w/v%, dextran 1.19w/v%, glucose 1.0w/v%, heparin sodium 625U/ml, the rest For medical injection;

[0066] B solution (100ml): 10w/v% human albumin injection

[0067] 2) Mix liquid A and liquid B in a volume ratio of 3:2, and place in a refrigerator at 4°C for later use.

[0068] 3) Take the cultured and expanded NK cells into a centrifuge tube and centrifuge at 300g for 10 minutes. Wash twice with PBS and count.

[0069] 4) According to the counting results, resuspend the NK cells with the pre-chilled cryopreservation solution prepared in 2) so that the cell density is 1×10 8 /ml, transfer the cells to the freezer bag.

[0070] 5) Place the cryopreservation bag containing the cells in a refrigerator at 4 degrees to equilibrate for 15 minutes.

[0071] 6) Place the cryopreservation bag containing the cells in the refrigerator at -80°C overnight.

[0072] 6) Store the cryopreservation bag containing the cells in liquid nitrogen.

[0073] 7) After 1, 6, and 12 months, remove the cells from the liquid nitrogen tank, place them in a 37°C water bath to thaw, and take a small amount of cells to detect the cell viability by trypan blue staining.

[0074] At the same time, the control freezing solution (50% FBS+40% RPMI1640+10% DMSO) was used to treat NK cells as a control, except for the freezing solution, the other steps were the same as above.

[0075] 3. Results

[0076] The comparison of NK resuscitation rates with different cryopreservation solutions is shown in Table 2:

[0077] Table 2 Comparison of NK cell resuscitation rates with different cryopreservation solutions

[0078] Example 2 Example 2 Control 1 month survival rate 96.4％ 82.6％ 6-month survival rate 92.8％ 75.3％ 12 months live rate 90.5％ 60.2％

Example Embodiment

[0079] Example 3: Preparation and application of immune cell cryopreservation solution for direct intravenous infusion

[0080] 1. Raw materials and specifications

[0081] Hydroxyethyl starch and sodium chloride injection: 500ml: Hydroxyethyl starch (200/0.5) 30g

[0082] Dextran 40 glucose injection: 500ml: 30g dextran 40 and 25g glucose

[0083] Heparin Sodium Injection: 2ml: 12,500 units.

[0084] Human albumin injection: 50ml: 5g total protein

[0085] 2. Preparation and application of immune cell cryopreservation solution for direct intravenous infusion

[0086] 1) Prepare liquid A and liquid B separately

[0087] A solution (100ml): 9ml dimethyl sulfoxide + 33ml hydroxyethyl starch sodium chloride injection + 57.9ml dextran glucose injection + 0.1ml heparin sodium injection.

[0088] The content of each component in A solution is: dimethyl sulfoxide 9v/v%, hydroxyethyl starch 1.98w/v%, dextran 3.47w/v%, glucose 2.9w/v%, heparin sodium 625U/ml, the rest For medical injection;

[0089] Solution B (100ml): 10w/v% human albumin injection.

[0090] 2) Mix liquid A and liquid B at a ratio of 3:2 and place in a refrigerator at 4°C for later use.

[0091] 3) Take the cultured and expanded CAR-T cells into a centrifuge tube, and centrifuge at 300g for 10 minutes. Wash twice with PBS and count.

[0092] 4) According to the counting results, resuspend CAR-T cells with the pre-chilled cryopreservation solution prepared in 2) so that the cell density is 5×10 7 /ml, transfer the cells to the freezer bag.

[0093] 5) Place the cryopreservation bag containing the cells in a refrigerator at 4°C for 15 minutes.

[0094] 6) Place the cryopreservation bag containing the cells in the refrigerator at -80°C overnight.

[0095] 6) Store the cryopreservation bag containing the cells in liquid nitrogen.

[0096] 7) After 1, 6, and 12 months, remove the cells from the liquid nitrogen tank, place them in a 37°C water bath to thaw, and take a small amount of cells to detect the cell viability by trypan blue staining.

[0097] At the same time, CAR-T cells treated with the control cryopreservation solution (50% FBS+40% RPMI1640+10% DMSO) were used as the control, except for the cryopreservation solution, the other steps were the same as above.

[0098] 3. Results

[0099] The comparison of CAR-T resuscitation rate with different cryopreservation solutions is shown in Table 3:

[0100] Table 3 Comparison of CAR-T cell resuscitation rate with different cryopreservation solutions

[0101] Example 3 Example 3 Control 1 month survival rate 98.1％ 85.5％ 6-month survival rate 96.7％ 80.5％ 12 months live rate 93.3％ 72.4％

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Description & Claims & Application Information

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Classification and recommendation of technical efficacy words

Avoid pollution
Good freezing effect

Direct venous re-transfusion immune cell cryopreservation medium and application thereof

AI-Extracted Technical Summary

Problems solved by technology

Abstract

Examples

Example Embodiment

Example Embodiment

Example Embodiment

PUM

Description & Claims & Application Information

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