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Method for preparing placenta hematopoietic stem cell preparation

A technology of hematopoietic stem cells and preparations, applied in the field of biomedicine, can solve the problems of low cell separation efficiency and inability to use directly, and achieve the effect of improving separation efficiency

Active Publication Date: 2015-07-01
HANGZHOU S EVANS BIOSCI LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, experiments have confirmed that in the above method, the isolation efficiency of placental cells is low, and placental hematopoietic stem cells must be resuscitated and cultured after cryopreservation, and cannot be used directly

Method used

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  • Method for preparing placenta hematopoietic stem cell preparation
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Embodiment approach

[0015] According to the present invention, wherein, according to a particularly preferred embodiment of the present invention, the enzyme solution contains 200-300 IU / mL type I collagenase, 30-50 IU / mL DNase I, 1.5-3.5 IU / mL dispase and 600-800IU / mL hyaluronidase; the freezing liquid contains 35-50mL / L dimethyl sulfoxide, 10-20g / L hydroxyethyl starch, 50-100g / L Human serum albumin, 10-20g / L dextran, 5-5.5g / L sodium chloride, 4.5-5g / L sodium gluconate, 3.4-3.9g / L sodium acetate, 0.3-0.45g / L Potassium chloride and 0.2-0.4g / L magnesium chloride. In this preferred embodiment, the present invention can further improve the separation efficiency of placental cells, and improve the hematopoietic colonization ability of frozen placental hematopoietic stem cells after returning to normal temperature.

[0016] According to the present invention, wherein, the weight ratio of the placental tissue and the enzyme solution can be 1: (0.5-5), preferably 1: (1-4); The volume ratio of the solu...

Embodiment 1

[0028] Digest the placental tissue with an enzyme solution at a temperature of 37°C for 30 minutes to obtain a digested product and filter through double-layer gauze to remove tissue residues in the digested product to obtain an enzymatic hydrolyzate; mix the enzymatically hydrolyzed solution with hydroxyethyl starch solution And let it stand for 20 minutes to separate the mixed materials to obtain the upper nucleated cell suspension; mix the nucleated cell suspension with the cryopreservation solution to obtain the placental hematopoietic stem cell preparation of this embodiment.

[0029] Wherein, the enzyme solution contains 250IU / mL type I collagenase, 40IU / mL DNase I, 2.4IU / mL dispase and 700IU / mL hyaluronidase; L of dimethyl sulfoxide, 15g / L of hydroxyethyl starch, 80g / L of human serum albumin, 15g / L of dextran, 5.2g / L of sodium chloride, 4.8g / L of sodium gluconate, 3.7g / L of sodium acetate, 0.40g / L of potassium chloride and 0.3g / L of magnesium chloride. The weight ratio...

Embodiment 2

[0031] Digest the placental tissue with an enzyme solution at a temperature of 37°C for 30 minutes to obtain a digested product and filter through double-layer gauze to remove tissue residues in the digested product to obtain an enzymatic hydrolyzate; mix the enzymatically hydrolyzed solution with hydroxyethyl starch solution And let it stand for 20 minutes to separate the mixed materials to obtain the upper nucleated cell suspension; mix the nucleated cell suspension with the cryopreservation solution to obtain the placental hematopoietic stem cell preparation of this embodiment.

[0032] Wherein, the enzyme solution contains 200IU / mL type I collagenase, 50IU / mL DNase I, 1.5IU / mL dispase and 800IU / mL hyaluronidase; L of dimethyl sulfoxide, 10g / L of hydroxyethyl starch, 100g / L of human serum albumin, 20g / L of dextran, 5g / L of sodium chloride, 5.0g / L of sodium gluconate, 3.4 g / L sodium acetate, 0.30g / L potassium chloride and 0.4g / L magnesium chloride. The weight ratio of the p...

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Abstract

The invention provides a method for preparing a placenta hematopoietic stem cell preparation. The method comprises the following steps: (1) digesting placenta tissues with an enzyme solution so as to obtain a digestive product and removing tissue residues in the digestive product to obtain enzymatic hydrolysate; (2) mixing the enzymatic hydrolysate with a hydroxyethyl starch solution, and layering the mixed materials to obtain a nuclear cell suspension; and (3) mixing the nuclear cell suspension with a frozen stock solution, wherein the enzyme solution contains type I collagenase, DNA enzyme I, dispase and hyaluronidase, and the frozen stock solution contains dimethyl sulfoxide, hydroxyethyl starch, human albumin and dextran. According to the technical scheme, the separation efficiency of the placenta hematopoietic stem cell can be improved; and the frozen placenta hematopoietic stem cell can be directly applied to stem cell treatment when heated to a room temperature.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for preparing a placental hematopoietic stem cell preparation. Background technique [0002] Placental stem cells are stem cells derived from placental tissue of newborns, including placental subtotipotent stem cells, placental hematopoietic stem cells, placental mesenchymal stem cells, placental maternal stem cells, etc. Placental hematopoietic stem cells are a group of primitive hematopoietic cells that exist in the placenta tissue. Hematopoietic stem cells are the progenitor cells of blood cells (red blood cells, white blood cells, platelets, etc.) and are highly undifferentiated cells. [0003] Studies of the human placenta have found that the placenta is a hematopoietic organ. From the third week of pregnancy until birth, the placenta not only performs functions such as material exchange, immune regulation, and secretion of active factors, but also undertakes h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/50
Inventor 杜小春陈锦阳李程项春生
Owner HANGZHOU S EVANS BIOSCI LTD
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