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A method for extracting umbilical cord blood hematopoietic stem cells under hypoxic environment

A technology for hematopoietic stem cells and umbilical cord blood is applied in the field of extraction of hematopoietic stem cells from umbilical cord blood. Effect

Active Publication Date: 2020-08-18
希瑞干细胞科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there are deficiencies in the current use of umbilical cord blood in clinical practice. The most critical technical bottleneck is the limited amount of umbilical cord blood and the small number of cells. In addition, normal hematopoietic stem cells reside in the hypoxic environment of bone marrow, umbilical cord blood, and placenta. , but almost all research on hematopoietic stem cells is carried out in non-physiological conditions of ambient air (ie, normoxia), including the isolation and extraction of hematopoietic stem cells, however, exposure to ambient air will cause non-physiological oxygen stress ( EPHOSS, extraphysiologic oxygen shock / stress), when the intracellular oxygen concentration suddenly increases, the intracellular reactive oxygen species (ROS) increase, and normal cells respond to oxidative stress, resulting in the accumulation of P53 in the mitochondrial matrix, triggering mitochondrial permeability The opening of the sex transition pore (mPTP) causes the electrochemical gradient of the membrane to collapse, and the apoptotic factors are released into the cytoplasm, eventually leading to cell apoptosis, which will reduce the cell yield in the collection and expansion of hematopoietic stem cells, and at the same time promote the differentiation of stem cells
In summary, the number of hematopoietic stem cells collected by traditional methods is seriously lower than the actual content, and the originality is low, which greatly limits its application in adult transplantation

Method used

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  • A method for extracting umbilical cord blood hematopoietic stem cells under hypoxic environment
  • A method for extracting umbilical cord blood hematopoietic stem cells under hypoxic environment
  • A method for extracting umbilical cord blood hematopoietic stem cells under hypoxic environment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 Experimental preparation

[0037] 1.1 Setup of hypoxic workstation

[0038]The schematic diagram of the hypoxic workstation is as follows: figure 1 shown. The hypoxic workstation (purchased from Ruskinn) simulates the internal environment of organisms, can precisely control the gas concentration, temperature and humidity in the station, and has an ISO4 grade HEPA filtration system, reaching the tenth grade of cleanliness. The working area of ​​the hypoxic workstation is divided into two left and right consoles (left console 1, right console 2), and a transfer box 3 in the middle. The material transfer between the two consoles and the materials between the console and the outside world Transfers can all be done via transfer boxes.

[0039] All aseptic operations in this experiment were carried out in the hypoxic workstation through the operation port 5. The hypoxic workstation was turned on 3 hours before the experiment, and the oxygen concentration was s...

Embodiment 2

[0048] The collection of embodiment 2 umbilical cord blood

[0049] A sterile blood collection bag containing anticoagulant was prepared before delivery, and the umbilical cord blood was collected by puncturing the umbilical cord vein immediately after the fetus was delivered. Cord blood collection was completed within 2 minutes of fetal delivery. The collection of umbilical cord blood exceeds 60ml is qualified. Check the integrity of the collection bag to ensure no fluid leaks. The collected umbilical cord blood is placed in an incubator, and the temperature in the incubator is controlled at 2-10°C. transport to the laboratory. If it cannot be processed immediately, the umbilical cord blood can be temporarily stored in a medical preservation box at 4°C. Cord blood is processed within 24 hours of collection.

Embodiment 3

[0050] The processing of embodiment 3 umbilical cord blood

[0051] Schematic diagrams of the structure of the double bag and the blood plasma clip are as follows: figure 2 , image 3 shown.

[0052] 3.1 Transfer of umbilical cord blood: close all roller clamps 12 of the double bag. Use a 50ml sterile syringe to suck out the umbilical cord blood from the blood collection bag, and transfer it to the main bag 7 of the double bag through the liquid inlet tube 9 . Transfer the double bag to the right station. Pipette a corresponding volume of Hespan, and add it to the cord blood through the transfer tube 11 (cord blood volume: Hespan volume = 4:1), and shake it gently to mix well.

[0053] 3.2 Settling of umbilical cord blood: Hang the main bag 7 of the double bag vertically on the hook on the wall of the operating table through the hanging hole 4, let it stand for about 1 hour, and observe the settling effect of the umbilical cord blood; during the settling process, it can b...

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Abstract

The invention discloses a method for extracting umbilical cord blood hematopoietic stem cells under a low-oxygen environment. The method specifically comprises the following steps: adding 80ng / ml to 120ng / ml of ciclosporin A into an anticoagulant added into a sterile blood collection bag; adding 30ng / ml to 70ng / ml of the ciclosporin A into a hydroxyethyl starch solution sedimentation agent; carrying out a whole-process extraction process of the hematopoietic stem cells in a sealed low-oxygen working station. The low-oxygen working station is provided with independent operation platforms at two sides and a transferring gate; the oxygen concentration of the operation platforms at two sides is set to 3 percent to 8 percent; the temperature of the operation platform at one side is set to 3 DEG C to 10 DEG C and the operation platform is used for carrying out preparation and sub-packaging of a reagent, transferring umbilical cord blood and cryopreserving the umbilical cord blood hematopoietic stem cells; the temperature of the operation platform at the other side is set to 18 DEG C to 25 DEG C and the operation platform is used for carrying out sedimentation of the umbilical cord blood, carrying out blood serum separation on the umbilical cord blood and centrifuging after the blood serum separation. According to the method disclosed by the invention, the hematopoietic stem cells with more quantity, more original state and strong proliferation and differentiation capabilities can be extracted.

Description

technical field [0001] The invention relates to a method for extracting umbilical cord blood hematopoietic stem cells, in particular to a method for extracting umbilical cord blood hematopoietic stem cells in a hypoxic environment. Background technique [0002] Hematopoietic stem cells (HSCs) are a small group of hematopoietic precursor cells with high self-renewal and multilineage differentiation potential. It has been confirmed that HSC can differentiate into at least 12 series of blood cells and other cells, including red blood cells, granulocytes, monocytes, eosinophils, basophils, mast cells, platelets, total lymphocytes, B lymphocytes, Dendritic cells, natural killer cells, and osteoblasts and fibroblasts in the bone marrow. Therefore, hematopoietic stem cell transplantation is an important means of treating malignant hematological diseases and certain immune diseases. At present, hematopoietic stem cells used in clinical practice mainly come from bone marrow and umb...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0789
CPCC12N5/0647C12N2500/02C12N2500/12C12N2500/34C12N2501/91
Inventor 朱丰城龚霄翔王浩楚晓婷
Owner 希瑞干细胞科技有限公司
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