Amniotic mesenchymal stem cell resuscitation culture medium and resuscitation culture method thereof

Abstract

The invention relates to the technical field of cell culture, particularly an amniotic mesenchymal stem cell resuscitation culture medium and a resuscitation culture method thereof. The preparation method of the resuscitation culture medium comprises the following steps: taking amniotic mesenchymal stem cells, carrying out first culture by using a complete culture medium, carrying out second culture by using a serum-free culture medium, and collecting the supernatant to obtain the resuscitation culture medium. When being used for amniotic mesenchymal stem cell resuscitation culture, the amniotic mesenchymal stem cell resuscitation culture medium is beneficial to amniotic mesenchymal stem cell resuscitation activity enhancement and cell proliferation, and can better maintain the biological characteristics of the amniotic mesenchymal stem cells.

Description

technical field

[0001] The invention relates to the technical field of cell culture, in particular to a recovery medium for amniotic mesenchymal stem cells and a recovery culture method thereof. Background technique

[0002] Neurodegenerative disease (Neurodegenerative disease) is a kind of disease that is caused by the blockage of the central nervous system, resulting in the loss of neuron cell function, thereby inhibiting the function of the central nervous system. It has the characteristics of high morbidity and mortality, mainly including : Stroke, traumatic brain injury, spinal muscular atrophy, amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease and Parkinson's disease, etc.

[0003] Neurodegenerative diseases all cause more severe symptoms. Among them, cerebral apoplexy is a disease in which acute cerebral blood circulation disorder is caused by stenosis, occlusion or rupture of intracerebral arteries caused by various predisposing factors. It is...

Examples

Embodiment 1

[0048] (1) Culture of amniotic mesenchymal stem cells and collection of conditioned medium

[0049] Shred the amnion tissue to 1.0-20.0cm 3 Finally, the tissue block was digested with 0.1% trypsin for 60 minutes, and then digested with 0.5% type I collagenase. When the tissue block was basically digested, it was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, resuspended with PBS, and passed through a 70 μm cell sieve. Centrifuge again at 2000 rpm for 5 min, and culture and resuspend the cell pellet with DMEM / F12+10% FBS. Place at 37°C, 5% CO 2 Culture the incubator statically, change the medium every 2-3 days, and when the confluence of the cell clones reaches 80%-90%, digest the cells with 0.25% trypsin, centrifuge and resuspend the pellet with complete medium, 7×10 3 / cm 2 The cell density was seeded in a culture dish to continue culturing. The P3-P5 generation cells were collected for conditioned medium collection.

[0050] Conditioned medium col...

Embodiment 2

[0065] (1) Culture of amniotic mesenchymal stem cells and collection of conditioned medium

[0066] Shred the amnion tissue to 1.0-20.0cm 3 Finally, the tissue block was digested with 0.1% trypsin for 60 minutes, and then digested with 0.5% type I collagenase. When the tissue block was basically digested, it was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, resuspended with PBS, and passed through a 70 μm cell sieve. Centrifuge again at 2000 rpm for 5 min, and culture and resuspend the cell pellet with DMEM / F12+10% FBS. Place at 37°C, 5% CO 2 Culture the incubator statically, change the medium every 2-3 days, and when the confluence of the cell clones reaches 80%-90%, digest the cells with 0.25% trypsin, centrifuge and resuspend the pellet with complete medium, 7×10 3 / cm 2 The cell density was seeded in a culture dish to continue culturing. The P3-P5 generation cells were collected for conditioned medium collection.

[0067] Conditioned medium col...

Embodiment 3

[0080] (1) Culture of amniotic mesenchymal stem cells and collection of conditioned medium

[0081] Shred the amnion tissue to 1.0-20.0cm 3 Finally, the tissue block was digested with 0.1% trypsin for 60 minutes, and then digested with 0.5% type I collagenase. When the tissue block was basically digested, it was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, resuspended with PBS, and passed through a 70 μm cell sieve. Centrifuge again at 2000 rpm for 5 min, and culture and resuspend the cell pellet with DMEM / F12+10% FBS. Place at 37°C, 5% CO 2 Culture the incubator statically, change the medium every 2-3 days, and when the confluence of the cell clones reaches 80%-90%, digest the cells with 0.25% trypsin, centrifuge and resuspend the pellet with complete medium, 7×10 3 / cm 2 The cell density was seeded in a culture dish to continue culturing. The P3-P5 generation cells were collected for conditioned medium collection.

[0082] Conditioned medium col...

technical field

[0001] The invention relates to the technical field of cell culture, in particular to a recovery medium for amniotic mesenchymal stem cells and a recovery culture method thereof. Background technique

[0002] Neurodegenerative disease (Neurodegenerative disease) is a kind of disease that is caused by the blockage of the central nervous system, resulting in the loss of neuron cell function, thereby inhibiting the function of the central nervous system. It has the characteristics of high morbidity and mortality, mainly including : Stroke, traumatic brain injury, spinal muscular atrophy, amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease and Parkinson's disease, etc.

[0003] Neurodegenerative diseases all cause more severe symptoms. Among them, cerebral apoplexy is a disease in which acute cerebral blood circulation disorder is caused by stenosis, occlusion or rupture of intracerebral arteries caused by various predisposing factors. It is...

Examples

Embodiment 1

[0048] (1) Culture of amniotic mesenchymal stem cells and collection of conditioned medium

[0049] Shred the amnion tissue to 1.0-20.0cm 3 Finally, the tissue block was digested with 0.1% trypsin for 60 minutes, and then digested with 0.5% type I collagenase. When the tissue block was basically digested, it was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, resuspended with PBS, and passed through a 70 μm cell sieve. Centrifuge again at 2000 rpm for 5 min, and culture and resuspend the cell pellet with DMEM / F12+10% FBS. Place at 37°C, 5% CO 2 Culture the incubator statically, change the medium every 2-3 days, and when the confluence of the cell clones reaches 80%-90%, digest the cells with 0.25% trypsin, centrifuge and resuspend the pellet with complete medium, 7×10 3 / cm 2 The cell density was seeded in a culture dish to continue culturing. The P3-P5 generation cells were collected for conditioned medium collection.

[0050] Conditioned medium col...

Embodiment 2

[0065] (1) Culture of amniotic mesenchymal stem cells and collection of conditioned medium

[0066] Shred the amnion tissue to 1.0-20.0cm 3 Finally, the tissue block was digested with 0.1% trypsin for 60 minutes, and then digested with 0.5% type I collagenase. When the tissue block was basically digested, it was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, resuspended with PBS, and passed through a 70 μm cell sieve. Centrifuge again at 2000 rpm for 5 min, and culture and resuspend the cell pellet with DMEM / F12+10% FBS. Place at 37°C, 5% CO 2 Culture the incubator statically, change the medium every 2-3 days, and when the confluence of the cell clones reaches 80%-90%, digest the cells with 0.25% trypsin, centrifuge and resuspend the pellet with complete medium, 7×10 3 / cm 2 The cell density was seeded in a culture dish to continue culturing. The P3-P5 generation cells were collected for conditioned medium collection.

[0067] Conditioned medium col...

Embodiment 3

[0080] (1) Culture of amniotic mesenchymal stem cells and collection of conditioned medium

[0081] Shred the amnion tissue to 1.0-20.0cm 3 Finally, the tissue block was digested with 0.1% trypsin for 60 minutes, and then digested with 0.5% type I collagenase. When the tissue block was basically digested, it was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, resuspended with PBS, and passed through a 70 μm cell sieve. Centrifuge again at 2000 rpm for 5 min, and culture and resuspend the cell pellet with DMEM / F12+10% FBS. Place at 37°C, 5% CO 2 Culture the incubator statically, change the medium every 2-3 days, and when the confluence of the cell clones reaches 80%-90%, digest the cells with 0.25% trypsin, centrifuge and resuspend the pellet with complete medium, 7×10 3 / cm 2 The cell density was seeded in a culture dish to continue culturing. The P3-P5 generation cells were collected for conditioned medium collection.

[0082] Conditioned medium col...

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