Human placenta, umbilical cord mesenchyma stemcell stock and its construction method

Abstract

The invention discloses a human placenta, umbilical cord mesenchyme stem cell bank and its making method. The method includes the following steps: the first is taking human placenta, umbilical cord to test; smashing after cleaning by phosphate buffer; adding phosphate buffer to dilute; the second is adding collagenase to digest; the third is adding phosphate buffer; the fourth is adding trypsinization; the fifth is mixing the cell from the second and fourth, centrifuging, removing supernatant, cleaning by the phosphate buffer, centrifuging, and removing supernatant; the mesenchyme stem cell are gained; the sixth is freezing them in liquid nitrogen; saving by ABO / Rh subtype and HLA subtype; setting recallable cell information file. The invention is newborn storage placenta and umbilical cord mesenchyme stem cell. It can offer mesenchyme stem cell to treat the disease for personal, family and others.

Description

technical field

[0001] The invention relates to a stem cell bank and a construction method thereof, in particular to a human placenta and umbilical cord mesenchymal stem cell bank and a construction method thereof. technical background:

[0002] Mesenchymal stem cells (MSCs) are a type of stem cells with two important characteristics of stem cells: strong self-proliferation ability and multi-differentiation potential. MSCs originated from mesoderm, and theoretically, it can differentiate to other mesoderm tissues. Recent studies have shown that under suitable in vivo or in vitro conditions, MSCs can not only differentiate into mesoderm mesenchymal tissue, but also maintain the differentiation potential of endoderm, and can differentiate into nerve cells, epithelial cells, cardiomyocytes, and osteoblasts. MSCs are co-cultured with fetal mouse midbrain or striatal cells, which can differentiate into neurons and glial cells, and MSCs are transplanted around the myocardial isch...

Examples

Embodiment 1

[0032] Preparation of human placental umbilical cord mesenchymal stem cells:

[0033] (1) After confirming the safety of human placenta and umbilical cord through strict testing procedures (ABO / Rh blood type testing, HLA typing testing, syphilis antibody testing, HIV immune testing, CMV antibody testing, hepatitis B antigen antibody testing, etc.), Rinse repeatedly with PBS (phosphate buffer solution) with a pH of 7.2 to remove residual blood, and cut it to about 1mm with a sterile knife 3 -1.5mm 3 small pieces or use other sterile tools to crush it into minced meat (1mm 3 -1.5mm 3 ), take 25ml of minced meat, add PBS with a pH of 7.2 to 50ml;

[0034] (2) Add 20ml of collagenase phosphate buffer solution with a mass / volume ratio of 0.1%, put it at 37°C for digestion for 1 hour, and continuously magnetically stir the mixture; filter the mixture with a 100-mesh screen, and filter Separate the cell suspension from the undigested tissue, place the filtered cell suspension in ...

Embodiment 2

[0044] (1) After confirming the safety of human placenta and umbilical cord through strict testing procedures (ABO / Rh blood type testing, HLA typing testing, syphilis antibody testing, HIV immune testing, CMV antibody testing, hepatitis B antigen antibody testing, etc.), Rinse repeatedly with PBS with a pH of 7.4 to remove residual blood, and cut it to about 1mm with a sterile knife 3 -1.5mm 3 small pieces or use other sterile tools to crush it into minced meat (1mm 3 -1.5mm 3 ), take 20ml of minced meat, add PBS with a pH of 7.4 to 50ml;

[0045] (2) Add 25ml of collagenase phosphate buffer solution with a mass / volume ratio of 0.05%, put it at 37°C for digestion for 70 minutes, and continuously magnetically stir the mixture; filter the mixture with a 200-mesh sieve, and filter Separate the cell suspension from the undigested tissue, place the filtered cell suspension in a high-speed centrifuge at 1500 rpm, centrifuge for 20 minutes, discard the supernatant, and shake the p...

Embodiment 3

[0052] (1) After confirming the safety of human placenta and umbilical cord through strict testing procedures (ABO / Rh blood type testing, HLA typing testing, syphilis antibody testing, HIV immune testing, CMV antibody testing, hepatitis B antigen antibody testing, etc.), Rinse repeatedly with PBS (pH 7.4) to remove residual blood, and cut it to about 1mm with a sterile knife 3 -1.5mm 3 small pieces or use other sterile tools to crush it into minced meat (1mm 3 -1.5mm 3 ), take minced meat 25ml, add PBS (pH is 7.4) to 50ml;

[0053] (2) Add 16.7ml of collagenase phosphate buffer solution with a mass / volume ratio of 0.2%, put it at 37°C for digestion for 40 minutes, and continuously magnetically stir the mixture; filter the mixture with a 100-mesh sieve, and filter Separate the filtered cell suspension from the undigested tissue, place the filtered cell suspension in a high-speed centrifuge at 2500 rpm, centrifuge for 10 minutes, discard the supernatant, and place the precipi...

technical field

[0001] The invention relates to a stem cell bank and a construction method thereof, in particular to a human placenta and umbilical cord mesenchymal stem cell bank and a construction method thereof. technical background:

[0002] Mesenchymal stem cells (MSCs) are a type of stem cells with two important characteristics of stem cells: strong self-proliferation ability and multi-differentiation potential. MSCs originated from mesoderm, and theoretically, it can differentiate to other mesoderm tissues. Recent studies have shown that under suitable in vivo or in vitro conditions, MSCs can not only differentiate into mesoderm mesenchymal tissue, but also maintain the differentiation potential of endoderm, and can differentiate into nerve cells, epithelial cells, cardiomyocytes, and osteoblasts. MSCs are co-cultured with fetal mouse midbrain or striatal cells, which can differentiate into neurons and glial cells, and MSCs are transplanted around the myocardial isch...

Examples

Embodiment 1

[0032] Preparation of human placental umbilical cord mesenchymal stem cells:

[0033] (1) After confirming the safety of human placenta and umbilical cord through strict testing procedures (ABO / Rh blood type testing, HLA typing testing, syphilis antibody testing, HIV immune testing, CMV antibody testing, hepatitis B antigen antibody testing, etc.), Rinse repeatedly with PBS (phosphate buffer solution) with a pH of 7.2 to remove residual blood, and cut it to about 1mm with a sterile knife 3 -1.5mm 3 small pieces or use other sterile tools to crush it into minced meat (1mm 3 -1.5mm 3 ), take 25ml of minced meat, add PBS with a pH of 7.2 to 50ml;

[0034] (2) Add 20ml of collagenase phosphate buffer solution with a mass / volume ratio of 0.1%, put it at 37°C for digestion for 1 hour, and continuously magnetically stir the mixture; filter the mixture with a 100-mesh screen, and filter Separate the cell suspension from the undigested tissue, place the filtered cell suspension in ...

Embodiment 2

[0044] (1) After confirming the safety of human placenta and umbilical cord through strict testing procedures (ABO / Rh blood type testing, HLA typing testing, syphilis antibody testing, HIV immune testing, CMV antibody testing, hepatitis B antigen antibody testing, etc.), Rinse repeatedly with PBS with a pH of 7.4 to remove residual blood, and cut it to about 1mm with a sterile knife 3 -1.5mm 3 small pieces or use other sterile tools to crush it into minced meat (1mm 3 -1.5mm 3 ), take 20ml of minced meat, add PBS with a pH of 7.4 to 50ml;

[0045] (2) Add 25ml of collagenase phosphate buffer solution with a mass / volume ratio of 0.05%, put it at 37°C for digestion for 70 minutes, and continuously magnetically stir the mixture; filter the mixture with a 200-mesh sieve, and filter Separate the cell suspension from the undigested tissue, place the filtered cell suspension in a high-speed centrifuge at 1500 rpm, centrifuge for 20 minutes, discard the supernatant, and shake the p...

Embodiment 3

[0052] (1) After confirming the safety of human placenta and umbilical cord through strict testing procedures (ABO / Rh blood type testing, HLA typing testing, syphilis antibody testing, HIV immune testing, CMV antibody testing, hepatitis B antigen antibody testing, etc.), Rinse repeatedly with PBS (pH 7.4) to remove residual blood, and cut it to about 1mm with a sterile knife 3 -1.5mm 3 small pieces or use other sterile tools to crush it into minced meat (1mm 3 -1.5mm 3 ), take minced meat 25ml, add PBS (pH is 7.4) to 50ml;

[0053] (2) Add 16.7ml of collagenase phosphate buffer solution with a mass / volume ratio of 0.2%, put it at 37°C for digestion for 40 minutes, and continuously magnetically stir the mixture; filter the mixture with a 100-mesh sieve, and filter Separate the filtered cell suspension from the undigested tissue, place the filtered cell suspension in a high-speed centrifuge at 2500 rpm, centrifuge for 10 minutes, discard the supernatant, and place the precipi...

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