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Kit for synchronously separating cord blood PRP, cord blood plasma and cord blood cells

A technology of blood cells and kits, applied in the field of simultaneous separation of cord blood PRP, cord blood plasma and cord blood cell kits, to achieve the effect of high purity, avoiding allergy risks and small residual rate

Active Publication Date: 2016-05-11
齐湘杰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Separation and isolation of umbilical cord blood PRP and umbilical cord blood plasma at the same time during the separation of umbilical cord blood cells has great clinical application value. There is no technology involving the simultaneous separation of the three components in the prior art. This kit meets the requirements of synchronous separation of umbilical cord blood PRP, Cord blood plasma and cord blood cells, filling the technical gap

Method used

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  • Kit for synchronously separating cord blood PRP, cord blood plasma and cord blood cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Preparation of kits for synchronous separation of umbilical cord blood PRP, umbilical cord blood plasma and umbilical cord blood cells

[0029] 1. Reagent preparation

[0030] The kit for isolating peripheral blood mononuclear cells of the present invention includes the following reagents:

[0031] (1) No. A liquid: diluent is PBS pH7.4±0.1, does not contain Ca 2+ and Mg 2+ , wherein 1-3% human albumin is added; wherein the preparation method of the PBS solution is as follows:

[0032] Preparation of mother liquor:

[0033] 0.2MNa 2 HPO 4 : Weigh 71.6gNa 2 HPO 4 -12H 2 O, dissolved in 1000ml water

[0034] 0.2MNaH 2 PO 4 : Weigh 31.2gNaH 2 PO 4 -2H 2 O, dissolved in 1000ml water

[0035] Prepare 0.2MPB first (pH=7.4, 100ml): Take 19ml of 0.2mol / L NaH 2 PO 4 , 81ml of 0.2mol / L Na 2 HPO 4 , you can.

[0036] Then dilute 0.2MPB (pH=7.4) as follows, that is, 0.01MPB (pH=7.4): take 50ml of 0.2MPB, add NaCl to 0.9% (g / 100ml), set the volume to 1...

Embodiment 2

[0042] Example 2: Influence of No. B Liquid Separation Liquid Density on Cord Blood Cell Separation Effect

[0043] Preparation of three kinds of density separation solutions: 40% polysucrose solution or powdered Fi-coll-400 was prepared into 9% polysucrose solution with Hank's solution, which was A solution with a density of 1.028g / ml; 60% compound diatrizoate Glumine injection is prepared into 34% solution with Hank's solution, is B liquid, and density is 1.198g / ml; Then according to formula: d=(dAVA+dBVB) / (VA+VB) (d and V are respectively the density of solution and volume), mix liquid A and liquid B according to the ratio of 50:18.29, 50:19.11, and 50:20.25 respectively, filter and sterilize with a 0.22 micron disposable filter, and prepare the densities of 1.073±0.001g / ml and 1.075 ±0.001g / ml, 1.077±0.001g / ml separation liquid. Take a cord blood of about 120ml, divide it into three parts on average, separate the cord blood cells with three separation fluids, and mark the...

Embodiment 3

[0047] Embodiment 3: the concrete description of the inventive method

[0048] Take 120ml of cord blood, divide it into three 50ml centrifuge tubes a, and centrifuge at 2500rpm for 20 minutes. The cord blood is obviously divided into two layers: the upper layer is plasma and a small amount of platelets, and the lower layer is red blood cells, granulocytes, and a large number of platelets. Use a 10ml pipette to draw the upper layer of plasma and a small amount of platelet layer into two 50ml centrifuge tubes b. Add 1.5 times the number A solution of "red blood cells, granulocytes and a large number of platelet layers" to the centrifuge tube a, mix well, carefully add it along the tube wall to 15 15ml centrifuge tubes that have previously added 7ml of solution B, and centrifuge at 2500rpm After 20 minutes, the cells in the centrifuge tube are divided into four layers from top to bottom. The first layer: liquid A and a large number of platelets, the second layer: a layer rich in ...

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Abstract

The invention aims at providing a kit for synchronously separating cord blood PRP, cord blood plasma and cord blood cells and a using method. The kit is adopted for synchronously separating the cord blood PRP, the cord blood plasma and the cord blood cells. By means of cord blood PRP two-part concentration, the concentration of blood platelets is increased by more than one time, and the number of red blood cells is small in order of magnitude; by means of plasma two-time purification, the cord blood plasma obtained through separation is high in purity and almost contains no red blood cell or blood platelet or mononuclear cell or granulocyte under microscopic examination; by means of cord blood cell two-time purification, the purity of the cord blood cells obtained through separation can reach 95% or above, the red cell and blood platelet residue rate is small, and the irritability risk caused by the components is greatly avoided. In the primary separation step of plasma, compared with a conventional separation technology, the using dosage of a separation reagent is reduced by one half in the cord blood cell separation step, and the separation cost is effectively reduced.

Description

technical field [0001] The invention belongs to the technical field of cell plasma separation, and in particular relates to a kit for synchronously separating umbilical cord blood PRP, umbilical cord blood plasma and umbilical cord blood cells and a use method. Background technique [0002] High-concentration platelet-rich plasma (Platelet-rich plasma, PRP) is a high-concentration platelet-rich plasma made from blood, which is generally prepared from autologous peripheral blood. Human platelets can produce a protein (fibrin, Chinese name: fibronectin, fibronectin) with cell adhesion function in a high concentration state, which can promote the secretion of a large number of platelets and promote wound, tissue healing and cell regeneration. Growth factors, so PRP is also called plasma rich in growth factors (plasma-richgrowthfactors, PRGFs)). PRP has the functions of rapid hemostasis, pain relief, and accelerated wound healing, and can greatly reduce the formation of postope...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2509/10
Inventor 齐湘杰王盛
Owner 齐湘杰
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