An optimized method for programmed cooling of umbilical cord blood hematopoietic stem cells

A technology of hematopoietic stem cells and umbilical cord blood, applied in the biological field, can solve the problems of program-controlled cooling, lack of control of the temperature of umbilical cord blood samples, and large differences in the quality of umbilical cord blood cryopreservation, so as to ensure contact and penetration, maintain proliferation ability, and good proliferation effect of ability

Active Publication Date: 2022-02-25
北京佳宸弘生物技术有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1) Lack of control over the temperature of the cord blood sample before adding the cryoprotectant; lack of control over the process of adding the cryoprotectant; lack of control over the contact time between the cord blood sample and the cryoprotectant (mainly DMSO); for optimal programming Lack of understanding of the starting temperature of cooling
[0006] 2) The setting of the key control points for controlling the phase change back temperature is not reasonable enough
[0008] 4) When freezing in batches, it is impossible to monitor the temperature of each sample of umbilical cord blood for program-controlled cooling, resulting in large differences in the frozen storage quality of different umbilical cord blood

Method used

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  • An optimized method for programmed cooling of umbilical cord blood hematopoietic stem cells
  • An optimized method for programmed cooling of umbilical cord blood hematopoietic stem cells
  • An optimized method for programmed cooling of umbilical cord blood hematopoietic stem cells

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Embodiment 1

[0040] This embodiment provides an optimized method for programmed cooling of umbilical cord blood hematopoietic stem cells, comprising the following steps:

[0041] S1, the cord blood after collection should be prepared and separated within 24 hours to remove red blood cells and plasma, and retain the nucleated cell components rich in hematopoietic stem cells, which is the cord blood sample; at the same time, the cord blood selected and to be frozen has similar The waste umbilical cord blood with nuclear cell count and hematocrit is prepared according to the same method as the umbilical cord blood sample and cryoprotectant is added, as a reference sample for subsequent program-controlled cooling;

[0042] S2, the cord blood sample and the cryoprotectant were pre-cooled at 4°C for 10 minutes, and then the cryoprotectant was added to the cord blood sample obtained in step 1) using a micro-injection pump, and the cord blood sample was added during the addition process Put it in ...

Embodiment 2

[0054]This embodiment is an improvement of the above-mentioned embodiment 1, and it is a refinement of the above-mentioned embodiment about the cryoprotectant. The cryoprotectant in this embodiment is a mixture of dimethyl sulfoxide and dextran 40, and the cryoprotectant The added volume was 1 / 4 of the volume of the cord blood sample; the cryoprotectant contained 50% (v / v) dimethyl sulfoxide (DMSO) and 5% (w / v) dextran 40.

[0055] Since DMSO is toxic to cells at room temperature, both the cord blood specimen and the cryoprotectant are pre-cooled in a 4°C refrigerator for 10 minutes in advance to ensure that both the cryoprotectant and the cord blood specimen are in a low temperature state when in contact, avoiding DMSO damage to cells.

[0056] Strictly control the addition process of the cryoprotectant. One is to use a micro-injection pump to ensure slow and uniform addition, and the control time is 10-15 minutes. The second is to ensure a low temperature environment of 0-4°...

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Abstract

The invention provides an optimized program-controlled cooling method for umbilical cord blood hematopoietic stem cells. The collected umbilical cord blood is prepared and separated, and a cryoprotectant is added to it under strict control. Program: Chamber at 3°C ​​for 3min; drop to specimen temperature -7°C or box to 11°C at a rate of 1°C / min; drop to box-50°C at a rate of 50°C / min; Rise to cabinet-25°C at a speed of 1°C / min and keep it at -25°C for 3 minutes; drop to specimen-50°C or cabinet-60°C at a speed of 1°C / min; drop to specimen-90°C at a speed of 10°C / min ℃ or box-120℃, transfer to liquid nitrogen for long-term storage. The invention effectively controls the addition process of the cryoprotectant and the initial temperature of the program-controlled cooling, and more accurately actively intervenes and controls the heat dissipation and temperature rise of the umbilical cord blood sample when it is frozen from a liquid to a solid state, thereby avoiding cryoprotection. It can better maintain the proliferation ability of umbilical cord blood hematopoietic stem / progenitor cells after thawing.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an optimized method for program-controlled cooling of umbilical cord blood hematopoietic stem cells. Background technique [0002] Umbilical cord blood hematopoietic stem cells can be used for hematopoietic stem cell transplantation to treat many diseases such as blood system and immune system, and has important clinical application value. Cord blood hematopoietic stem cells are mostly stored in cord blood banks in the form of liquid nitrogen cryopreservation, and then thawed and resuscitated in clinical application. The maintenance of cell activity and proliferation ability of umbilical cord blood hematopoietic stem cells after thawing is closely related to the cryopreservation process of umbilical cord blood. For routine cord blood cryopreservation, see figure 1 . [0003] Program-controlled cooling is the core of the cryopreservation process. Through program-control...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
CPCA01N1/0284A01N1/0221
Inventor 魏晓飞李红莉张昱
Owner 北京佳宸弘生物技术有限公司
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