Kit for allogeneic peripheral blood mononuclear cell separation in vitro and application method of kit

Abstract

The invention relates to a kit for high-purity high-recovery separation in vitro of allogeneic peripheral blood mononuclear cells and an application method of the kit. The allogeneic mononuclear cells separated by the kit can be used for immunotherapy of unexplained habitual abortion. The kit comprises a reagent I, namely a peripheral blood thinner, a reagent II, namely an erythrocyte sedimentation solution, a reagent III, namely mononuclear cell separating solution and a reagent IV, namely a mononuclear cell washing solution. The method for separating the allogeneic peripheral blood mononuclear cells by using the kit comprises the following steps: anticoagulation pre-centrifugation; erythrocyte initial reaction; erythrocyte sedimentation; two-step separation of the mononuclear cell separating solution; secondary purification of mononuclear cells; bacteria detection; mononuclear cell cryopreservation. According to the technology, the mononuclear cell recovery rate can achieve more than 90 percent, the purity can achieve more than 95 percent, the survival rate can achieve more than 98 percent, and the service efficiency of the peripheral blood mononuclear cells is greatly improved. Plasma is removed through the step of anticoagulation pre-centrifugation. Compared with the conventional separation technology, the dose of the separation reagent is reduced, and the separation cost is effectively reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a high-purity and high-recovery allogeneic peripheral blood mononuclear cell in vitro separation kit and a method of use. The allogeneic mononuclear cells separated by the kit can be used for immunotherapy for unexplained habitual abortion . The kit of the present invention includes reagent I: peripheral blood dilution solution, reagent II: erythrocyte sedimentation solution, reagent III: mononuclear cell layering solution, and reagent IV: mononuclear cell washing solution. Using the kit of the present invention to separate allogeneic peripheral blood mononuclear cells in vitro can achieve the following effects: ① The recovery rate of mononuclear cells can reach more than 90%, and the survival rate can reach more than 98%, which greatly improves the use efficiency of peripheral blood mononuclear cells; ②The purity of isolated peripheral blood mononuclear cells can reach mor...

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Problems solved by technology

However, the main problems in the prior art are the low purity of isolated mononuclear cells, and large residual red blood cells and platelets, which lead to an increased risk of allergic reactions after transfusion

However, the above method requires 72 h of incubation at 37 °C, which will inevitably increase the CO 2 Expensive instruments such as cell incubators are not conducive to popularization; moreover, the above-mentioned method is cumbersome to operate and unfavorable to clinical promotion; moreover, the above-mentioned scheme cannot effectively solve the problem of improving the separation purity and reducing the residual red blood cells or platelets

In addition, due to the impact of cell cryopreservation and resuscitation on activity, and the current domestic cell cryopreservation methods mostly use cryopreservation solutions composed of DMSO, animal serum and cell culture medium in different proportions, it is inevitable that due to the introduction of exogenous animal serum , resulting in the possibility of animal pathogen contamination, which will affect the application of allogeneic peripheral blood mononuclear cells in immunotherapy. If in order to avoid the above problems, adopt the method of collecting and administering immediately, although the above problems can be avoided , but due to the determination of treatment timing, collection time, collection cost and other issues, it is urgent to provide a method of mixing cryopreservation solution suitable for in vivo cell preparations in actual operation

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