Serum-free medium of immune cells

A technology of serum-free culture medium and immune cells, which is applied in the fields of biology and medicine, can solve the problems of not supporting the growth of immune cells well, the source of human AB serum is difficult, and the supply is limited, so as to achieve stable properties, reduce accidental infections, The effect of simple ingredients

Active Publication Date: 2015-02-25
EASTERN UNION STEM CELL & GENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The advantage of using animal serum is that there is enough supply and the price is cheap, but the disadvantage of using animal serum is the risk of potential transmission of infectious diseases, and the occurrence of allergies due to foreign proteins, and animal serum d

Method used

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  • Serum-free medium of immune cells
  • Serum-free medium of immune cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: the preparation of 1000ml serum-free medium

[0021] Mix one liter bag of IMDM dry powder (Gibco), 10g bovine serum albumin (Roche), 10mg fatty acid (sigma), 20mg cholesterol (sigma), 20mg insulin (sigma), 15mg transferrin (sigma), 5mg 2-mercapto Ethanol (Amresco), 434mg Propandipeptide (Sigma), 20mg Glycerides (Sigma), 3.024g NaHCO 3 (sigma); add ultrapure water to 1000ml, stir to dissolve, adjust the pH value to about 7.0, filter and sterilize with a 0.22μm filter membrane, and store at 4°C.

Embodiment 2

[0022] Example 2: Isolation of hematopoietic stem cells

[0023] Under sterile conditions, take 50-80ml of umbilical cord blood from full-term cesarean section or full-term normal pregnant women with negative hepatitis B virus test, put it in a blood collection bag containing CPDA1 compound anticoagulant, and store it at 4°C. All samples should be processed within 12 hours Separation; one portion of cord blood is divided into three. Each portion was diluted with normal saline 1:1, and the diluted umbilical cord blood was slowly added to the upper layer containing Ficoll Hypaque human lymphocyte separation medium along the tube wall (the ratio of the two was 1:1), and its relative density was 1.077g / L. Carry out gradient centrifugation (2000r / min×30), take the cloudy mononuclear cell layer in the middle, centrifuge and wash twice with IMDM culture medium, each time at 1000r / min, and centrifuge for 5min. After discarding the supernatant, resuspend the cells with serum-free medi...

Embodiment 3

[0026] Embodiment 3: CIK cell induction and testing

[0027] Under sterile conditions, take 50-80ml of umbilical cord blood from full-term cesarean section or full-term normal pregnant women with negative hepatitis B virus test, put it in a blood collection bag containing CPDA1 compound anticoagulant, and store it at 4°C. All samples should be processed within 12 hours Separation; one portion of cord blood is divided into three. Each portion was diluted with normal saline 1:1, and the diluted umbilical cord blood was slowly added to the upper layer containing Ficoll Hypaque human lymphocyte separation medium along the tube wall (the ratio of the two was 1:1), and its relative density was 1.077g / L. Carry out gradient centrifugation (2000r / min×30), take the cloudy mononuclear cell layer in the middle, centrifuge and wash twice with IMDM culture medium, each time at 1000r / min, and centrifuge for 5min. After discarding the supernatant, adjust the cells to 1×10 with IMDM medium 7...

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Abstract

The invention discloses a serum-free medium for culturing immune cells. The medium includes a basic medium, bovine serum albumin, fatty acids, cholesterols, insulin, transferrin, 2-mercaptoethanol, N-(2)-L-alanyl-L-glutamine and glyceride. The serum-free medium has the advantages of simple component, stable properties and high CIK induction efficiency, and CIK cells cultured through the medium have the advantages of small batch difference, stable quality, convenient quality control, and reduction of accidental infection of patients, and are of great significance to promoting and applying immunotherapy.

Description

technical field [0001] The invention relates to the fields of biology and medicine, in particular, the invention relates to a serum-free culture medium of immune cells. Background technique [0002] Adoptive immunotherapy (ACI) refers to infusing immune cells with anti-tumor activity into tumor patients to directly kill or stimulate the body's immune response to kill tumor cells, so as to achieve the purpose of treating tumors. It includes non-specifically activated and specifically activated effector cells. The former uses non-specific stimulators (IL2, interferon) to stimulate precursor effector cells to activate them into effector cells with anti-tumor activity, such as LAK cells, tumor infiltration Cytokine induced kill cells (CIK), etc.; specifically activated effector cells refer to anti-tumor effector cells induced by using tumor antigens as stimulants, such as dendritic cells (DC), cytotoxic T lymphocytes (CD8+ cells), etc. The application of ACI in the treatment o...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 胡少青刘洋赵静邱晔张丽丽吴明远王翔孙迎秋施洁琦范星洲
Owner EASTERN UNION STEM CELL & GENE ENG
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