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Preparation method of placental hematopoietic stem cells

A technology for hematopoietic stem cells and placenta, applied in the biological field, can solve the problems of easy loss of cells, complicated operation steps, long separation process, etc., and achieve the effects of avoiding cell contamination, improving the separation rate, and improving the recovery rate.

Active Publication Date: 2019-02-15
山东康华生物医疗科技股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But above-mentioned prior art has following shortcoming: (1) separation process is long, operation steps are loaded down with trivial details, easily lose cell while, very easily cause cell pollution; Controlling the progress of the digestion process can easily lead to over-digestion, inactivation due to cell damage, and the use of a large number of enzymes will inevitably increase the cost of the experiment
[0006] In addition, the cryopreservation solution currently used in the cryopreservation method of hematopoietic stem cells is supplemented with fetal bovine serum or the donor's own serum. The serum contains a large number of unknown components, and the quality is not easy to control. must be troubled

Method used

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  • Preparation method of placental hematopoietic stem cells
  • Preparation method of placental hematopoietic stem cells
  • Preparation method of placental hematopoietic stem cells

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Embodiment 1

[0032] A method for preparing placental hematopoietic stem cells, comprising the following steps:

[0033] (1) Placenta pretreatment: Under aseptic conditions, use placenta washing solution (per 1L, pH7.2 phosphate buffer solution containing 100mg / L penicillin, 100mg / L streptomycin) element, 1mg / mL vitamin C, 100mg / L gentamicin, 50mg / L amphotericin B) for cleaning and set aside;

[0034] (2) placenta perfusion once: get the placenta in step (1), placenta perfusate A (contain 100mg / L penicillin, 100mg / L streptomycin, 1mg / mL vitamin C, 1 vial of heparin sodium injection) into the umbilical vein along the placental umbilical artery, press the placenta during injection to make the perfusate infiltrate the blood vessels inside the placenta, and collect the liquid components after perfusion;

[0035] (3) Secondary perfusion of placenta: take the placenta in step (2), placenta perfusate B (contain 100mg / L penicillin, 100mg / L streptomycin, 1mg / mL vitamin C , 0.05wt% neutral protease...

Embodiment 2

[0041] A method for preparing placental hematopoietic stem cells, comprising the following steps:

[0042] (1) Placenta pretreatment: Under sterile conditions, freshly collected full-term healthy placenta was washed with placenta washing solution (each L of phosphate buffer contains 200 mg / L penicillin, 200 mg / L streptomycin, 2 mg / mL Vitamin C, 200mg / L gentamicin, 80mg / L amphotericin B) cleaning, standby;

[0043] (2) placenta perfusion once: get the placenta in step (1), placenta perfusate A (contain 200mg / L penicillin, 200mg / L streptomycin, 2mg / mL vitamin C, 2 injections of heparin sodium injection) into the umbilical vein along the placental umbilical artery, press the placenta during the injection process to make the perfusate infiltrate the blood vessels inside the placenta, and collect the liquid components after perfusion;

[0044] (3) Placental perfusion for the second time: get the placenta in step (2), placental perfusate B (contain 200mg / L penicillin, 200mg / L strep...

Embodiment 3

[0050] A method for preparing placental hematopoietic stem cells, comprising the following steps:

[0051] (1) Placenta pretreatment: Under aseptic conditions, freshly collected full-term healthy placenta was washed with placenta washing solution (each L phosphate buffer solution contains 300mg / L penicillin, 300mg / L streptomycin, 2mg / mL Vitamin C, 200mg / L gentamicin, 100mg / L amphotericin B) cleaning, standby;

[0052] (2) placenta perfusion once: get the placenta in step (1), placenta perfusate A (contain 300mg / L penicillin, 300mg / L streptomycin, 2mg / mL vitamin C, 2 injections of heparin sodium injection) into the umbilical vein along the placental umbilical artery, press the placenta during the injection process to make the perfusate infiltrate the blood vessels inside the placenta, and collect the liquid components after perfusion;

[0053] (3) Placenta secondary perfusion: get the placenta in step (2), placenta perfusate B (contain 300mg / L penicillin, 300mg / L streptomycin,...

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to a preparation method of placental hematopoietic stem cells. The preparation method comprises the following steps: (1) pretreatment of a placenta; (2) first lavage of the placenta; (3) second lavage of the placenta; (4) third lavage of the placenta; (5) mixing and centrifuging; (6) primary purification of the placenta;and (7) cryopreservation of hematopoietic stem cells. The preparation method can improve the separation quantity and cell activity of CD34 positive cells, the adopted reagents have no potential risks, and can be applied clinically, and the experimental cost is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing placental hematopoietic stem cells. Background technique [0002] Hematopoietic stem cells (HSCs) are adult stem cells in the blood system. They are a heterogeneous population with long-term self-renewal ability, that is, they can produce two cells with the same properties as before division after going through a cell cycle. , and has the potential to differentiate into various mature blood cells under certain external conditions. [0003] Hematopoietic stem cells are the first to have major pathogenic mutations such as chromosomal translocations. This mutation does not affect their differentiation ability and can differentiate into mature blood cells with normal functions. The second mutation occurs in the mature blood cells differentiated by translocated hematopoietic stem cells, which will cause leukemia, such as acute myeloid leukemia (AML) and...

Claims

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Application Information

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IPC IPC(8): C12N5/0789A01N1/02
CPCA01N1/0221C12N5/0647C12N2509/00
Inventor 杨致亭韩伟邱青芳姚立琼潘德芹
Owner 山东康华生物医疗科技股份有限公司
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