Method for amplifying natural killer cells from placenta tissue

A natural killer cell and tissue technology, applied in the field of bioengineering, can solve the problems of long extraction time, small number of mononuclear cells, and low purity

Active Publication Date: 2022-03-04
BOYALIFE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The current method of separating and extracting mononuclear cells from blood (such as placental blood, umbilical cord blood, and peripheral blood) is often artificially using a polysucrose solution with a density of 1.077g / ml. This method has many limitations, such as extraction takes too long Long time (mostly between 2 and 3 hours for a single experiment), the number of extracted mononuclear cells is small, and the purity is too low, which greatly affects the follow-up experiments, and there is also a need for improvement in this aspect

Method used

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  • Method for amplifying natural killer cells from placenta tissue
  • Method for amplifying natural killer cells from placenta tissue
  • Method for amplifying natural killer cells from placenta tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1: the acquisition of placental blood

[0080] 1. Placenta cleaning: Use surgical forceps to take out the placental tissue (50g), put it in a stainless steel tray, and wash the surface of the placenta with a small amount of tissue cleaning solution containing penicillin-streptomycin-amphotericin to remove blood coagulation stains on the surface [In the present invention, if not otherwise stated, the tissue cleaning solution used is to prepare the gained solution in the following way: be mixed with penicillin 100U / ml, streptomycin 0.1mg / ml and 0.25 with 0.9% sodium chloride injection The solution of μg / ml amphotericin B was sterilized by filtration to obtain];

[0081] 2. Use scissors and tweezers to bluntly peel off and discard the amniotic layer on the surface of the placenta, and cut off the umbilical cord tissue above it. Then use scissors to cut the remaining placental lobule tissue into 3-7cm 3 Take a 300-mesh stainless steel filter and place it on t...

Embodiment 2

[0082] Example 2: Digestion of Placental Lobules Tissue

[0083] 1. Configuration of digestive juice:

[0084] 1. Add PBS to the type I collagenase powder, fully mix and dissolve, and configure a type I collagenase solution with a concentration of 10 mg / ml; Salt buffer solution, its preparation method is: take 250ml of 0.2mol / L potassium dihydrogen phosphate solution, add 118ml of 0.2mol / L sodium hydroxide solution, dilute with water to 1000ml, shake well, and obtain];

[0085] ②Add HBSS buffer solution to the type II collagenase powder, fully mix and dissolve, and configure a type II collagenase solution with a concentration of 10 mg / mL; [In the present invention, unless otherwise specified, the HBSS buffer solution used is obtained by The resulting solution was prepared as follows: 8.0 g of NaCl, 0.4 g of KCl, 0.1 g of MgSO4 7H2O, 0.1 g of MgCl2 6H2O, 0.06 g of Na2HPO4 2H2O, 0.06 g of KH2PO4, 1.0 g of glucose, 0.14 g CaCl2, 0.35g of NaHCO3 add distilled water to 1000ml t...

Embodiment 3

[0092] Example 3: Using Extraction of single nucleocytes from placental blood and placental cell suspension by automated separation equipment cell

[0093]1. Mix the placental blood obtained in Example 1 and the placental lobular tissue cell suspension obtained in Example 2, and inject the mixed solution into the 200ml blood collection bag containing anticoagulant 3.2% sodium citrate solution with a sterile syringe. The volume ratio of the coagulant to the biological sample is 1:12, after mixing, place it on a shaker and shake it slowly for 15 minutes;

[0094] 2. will The plastic needle of the disposable separation cup of the automatic separation equipment is inserted into the sterile interface on the blood collection bag, and the blood collection bag is hung up, so that the blood in it flows naturally into the central compartment of the separation cup; the pipeline is welded with a sterile splicer Separate the blood collection bag from the disposable separation cup...

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Abstract

The invention provides a method for amplifying natural killer cells from placenta tissues, which comprises the following steps: (1) taking perinatal placenta tissues, and cutting down villus lobule structure tissues in a small block form; (2) digesting placental lobule tissues by utilizing I and II type collagenase, DNA enzyme and thermolysin; (3) extracting mononuclear cells in the digested cell suspension and mononuclear cells in placenta blood by using a full-automatic separation device method; (4) inducing amplification of the NK cells in the mononuclear cells by utilizing cell factors such as IL-15, FLT3-L and OK432; and (5) carrying out biological identification on the amplified NK cells. The method disclosed by the invention has excellent technical effects as described in the specification.

Description

technical field [0001] The invention relates to a method for culturing and amplifying natural killer cells from placental villi lobular tissue and placental blood-derived cells, belonging to the technical field of bioengineering. Background technique [0002] Natural killer cells (NK cells) were discovered in peripheral blood 30 years ago, and CD3-CD56+ lymphocytes were defined as human NK cells. NK cells usually contain a large amount of perforin and granzyme B. When activated NK cells encounter target cells, NK cells release perforin and granzyme B to attack target cells. NK cells can also secrete cytokines such as IFN-γ, TNF-α, GM-CSF, and IL-3, which can directly act on target cells or attack target cells by activating other types of immune cells. [0003] It has been reported in literature (Koepsell SA, Miller JS, McKenna DH Jr. Natural killer cells: a review of manufacturing and clinical utility. Transfusion. 2013, 53(2): 404-10) that although the number of NK cells i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/073C12N5/078C12N5/02
CPCC12N5/0646C12N5/0605C12N5/0634C12N2501/2315C12N2501/26C12N2500/72C12N2500/32C12N2500/24C12N2509/00C12N2509/10
Inventor 魏卿肖海蓉刘庆喜
Owner BOYALIFE
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