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Preparation method and preservation method of clinical application-level placental hematopoietic stem cells

A technology of hematopoietic stem cells and clinical application, which is applied in the field of preparation of clinical application-grade placental hematopoietic stem cells and preservation of placental hematopoietic stem cells. It can solve the problems of large processing capacity, patient infection, and maintenance, and achieve reduced digestive enzyme consumption and high cell activity. , handle large volume effects

Inactive Publication Date: 2014-05-14
台州恩源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prior art has the following defects: (1) The content of isolated placental hematopoietic stem cells is relatively low. For example, the number of mononuclear cells extracted in the patent "A new method for extracting hematopoietic stem cells from placental tissue for establishing a hematopoietic stem cell bank" is 2- 4×10 9 , the content of CD34-positive hematopoietic stem cells is only 0.3-0.7%, and the number of CD34-positive hematopoietic stem cells is at most 2.8×10 7 (2) The volume of the whole placenta or placental chorionic tissue is relatively large, so the processing volume is large, not only a large amount of digestive enzymes are used, which increases the cost, but also takes time, which is not conducive to the maintenance of cell activity
However, there is no serum suitable for clinical application at present. Serum contains a large number of unknown components, and the quality is not easy to control. There are safety hazards in allogeneic infusion. For example, serum contains incurable viruses or pathogens. very serious
Although albumin at a suitable concentration can maintain a high cell survival rate for a certain period of time, the albumin currently used is of human or animal origin, which has potential safety hazards and is not suitable for clinical application.

Method used

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  • Preparation method and preservation method of clinical application-level placental hematopoietic stem cells
  • Preparation method and preservation method of clinical application-level placental hematopoietic stem cells
  • Preparation method and preservation method of clinical application-level placental hematopoietic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Acquisition of placental hematopoietic stem cells

[0038] 1. Collect the placenta of normal vaginal delivery or caesarean section under aseptic conditions, pre-treat immediately, and then separate two umbilical arteries and one umbilical vein, and insert a puncture needle connected to a syringe or a constant-flow pump or a pediatric scalp needle into the umbilical cord vein. Use 300ml of DEME culture solution containing penicillin and streptomycin (the concentration of antibiotics is 200mg / L, and the ratio of penicillin and streptomycin added is 1:1) as the cleaning solution to perfuse the placental villi vessels, and collect the cleaning fluid after perfusion. liquid;

[0039]2. Enzyme digestion. In order to better illustrate the practicability of the present invention, three different digestion methods are listed here for specific description, but the enzyme digestion method of the present invention is not limited thereto:

[0040] Enzyme digestion method...

Embodiment 2

[0049] Example 2: Detection of placental hematopoietic stem cells

[0050] 1. After the pretreatment of the freshly delivered placenta and before the preparation of placental hematopoietic stem cells: Hepatitis virus and corresponding antibody detection, AIDS antibody detection, syphilis antibody detection;

[0051] 2. At the same time as placental hematopoietic stem cells are prepared: bacteria and fungi are tested;

[0052] 3. After the placental hematopoietic stem cells are prepared: Quantitative detection of hematopoietic stem cells (CD34) and activity of hematopoietic stem cells (trypan blue staining).

Embodiment 3

[0053] Example 3: Cryopreservation of placental hematopoietic stem cells

[0054] 1. Prepare cryopreservation solution (10% DMSO + 5% human recombinant albumin + DEME culture solution): take a centrifuge tube, add 9ml DEME culture solution, then slowly add 6.75ml DMSO dropwise to the centrifuge tube, while adding After oscillating and shaking well, cover the lid, tighten it, and place it in a refrigerator at 4°C for pre-cooling. After the pre-cooling is completed, slowly add 6.75ml human recombinant albumin (purchased from Mitsubishi Pharmaceutical Group Co., Ltd. of Japan) dropwise, and shake while adding Mix well to avoid precipitation, cover the lid, tighten it, and pre-cool in a 4°C refrigerator for later use;

[0055] 2. Take out the pre-prepared cryopreservation solution, slowly drop it into the cell suspension, add 10ml to each tube, shake while dropping, mix well quickly, cover the lid, and pre-cool in a 4°C refrigerator;

[0056] 3. Take out the pre-cooled cell suspe...

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Abstract

The invention provides a preparation method of clinical application-level placental hematopoietic stem cells. The preparation method comprises the steps of pretreating a fresh delivered placenta immediately and then separating out umbilical arteries and umbilical veins, pouring a cleaning fluid into the chorionic vessels of the placenta and colleting the cleaning fluid after pouring, pouring an enzyme digestion fluid into the chorionic vessels of the placenta and digesting at 37 DEG C for 5-20min, pouring the collected fluid into the placenta and collecting the liquid after pouring, centrifuging the obtained fluid and re-suspending cells after removing the supernatant, and separating the resuspended cell suspension through a two-step process with hydroxyethyl starch and a lymphocyte separation medium to obtain the clinical application-level placental hematopoietic stem cells. The method is capable of increasing the number and the activity of the prepared placental hematopoietic stem cells and the content of CD34 positive cells, and the prepared placental hematopoietic stem cells are at the clinical application level and free of potential pathogenic contamination, and moreover, the method is low in preparation cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing clinical application-grade placental hematopoietic stem cells and a method for preserving the prepared placental hematopoietic stem cells. Background technique [0002] The latest research shows that the placenta contains a large number of hematopoietic stem cells, the same as the hematopoietic stem cells in umbilical cord blood, [0003] Placental hematopoietic stem cells are highly undifferentiated cells, which can also be said to be the original cells of all blood cells (most of which are immune cells), and can be used to treat a variety of blood system diseases and immune system diseases, including hematological malignancies (such as leukemia , multiple myeloma, myelodysplastic syndrome, lymphoma, etc.), hemoglobinopathies, bone marrow hematopoietic failure (such as aplastic anemia), congenital metabolic diseases, congenital immunodeficiency diseases, autoi...

Claims

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Application Information

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IPC IPC(8): C12N5/0789A01N1/02
Inventor 陈正琳夏佳音陈平
Owner 台州恩源生物科技有限公司
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