CN103081892B - Frozen stock solution used for preserving adult stem cells for long time and preparation method thereof - Google Patents

Abstract

The invention relates to a frozen stock solution used for preserving adult stem cells for a long time, and a preparation method thereof. Every 100ml of base frozen stock solution contains: 10-20ng/ml of herba epimedii total flavones, and every 100ml of base frozen stock solution contains: 50-70% of DMEM (dulbecc' modified eagle medium) cluture medium, 10% of dimethyl sulfoxide, and 40-20% of Knock-out serum. The frozen stock solution can improve capacities for preventing cells from frozen damage and resisting oxidation and apoptosis after the cells are recovered, prevents the cells from contacting with heterologous animal original serum during a frozen stock process and further increases security of the adult stem cells for clinic applications.

Description

A kind of for long-term cryopreserving liquid of preserving adult stem cell and preparation method thereof

Technical field

The invention belongs to cellular system engineering field, relate to a kind of for long-term cryopreserving liquid of preserving adult stem cell and preparation method thereof.

Background technology

Barren wort total chromocor (EF) is the extract of Chinese medicine, can promote propagation, migration and the oxidation resistance of stem cell.Adopt biochip technology to find that EF has remarkable rise effect to cortisone rat growth hormone (GH), growth hormone releasing hormone (GHRH) and all kinds of somatomedin as insulin-like growth factor binding protein (IGFBP), nerve growth factor (NGF) etc.Naturally-aged rat (nephrasthenia syndrome model), find that EF can make the gene expression rejuvenation of a plurality of tissues, also make GH, the GHRH of senile rat downward and the up-regulated of IGFBP, NGF etc.Adopt the embryo mouse neural stem cell of in-vitro separation further to prove that barrenwort and extract thereof have directly short propagation, anti-apoptotic effect to stem cell.In addition damage has repair to Cold exposure mouse tissue, to have the antioxidation that report shows barren wort total chromocor.

At present, low temperature is preserved the protection that stem cell mainly relies on anti frozen liquid dimethyl sulfoxide (DMSO) (DMSO) (SIGMA, D5879) and serum.And dimethyl sulfoxide (DMSO) has damage to cell, and in recovery process, easily cause stem cell oxidative damage and differentiation potential to reduce.

Summary of the invention

In order to solve in existing cells frozen storing liquid, protect the problems such as composition is single; the present invention's application barren wort total chromocor (Xi'an grass roots biotechnology Co., Ltd; 20100806) be additive; with Knock-out serum (Gibco; 10828028) substitute conventional hyclone; improve anti-apoptosis, oxidation resistance after adult stem cell recovery, and remove the impact of external source animal sources serum on cell.

The present invention is by the following technical solutions:

1. one kind for the long-term cryopreserving liquid of preserving adult stem cell, it is characterized in that containing in the cryopreserving liquid of every 100ml basis: barren wort total chromocor 10～20ng/ml, and in the cryopreserving liquid of every 100ml basis, contain: DMEM medium 50%～70%, dimethyl sulfoxide (DMSO) 10%, Knock-out serum 40%～20%.

2. the preparation method of the cryopreserving liquid for long-term preservation adult stem cell of the 1st more than, comprises the following steps:

In dimethyl sulfoxide (DMSO): Knock-out serum: DMEM medium ratio is that 1:2:7 concentration is prepared basic cryopreserving liquid;

1mg barren wort total chromocor is added in the DMEM culture fluid of 10ml, be mixed with the mother liquor of 105ng/ml, then get mother liquor 1 μ l and be diluted in the above-mentioned basic cryopreserving liquid of 10ml, obtain the cryopreserving liquid that required final concentration is 10ng/ml.

Embodiment

The preparation of basis cryopreserving liquid:

Cryopreserving liquid preparation order: first add dimethyl sulfoxide (DMSO) in DMEM medium, then add Knock-out serum;

Configuration proportion: DMEM medium, dimethyl sulfoxide (DMSO), serum are prepared basic cryopreserving liquid in 7:1:2 ratio;

The preparation of barren wort total chromocor freezing liquid:

1mg barren wort total chromocor is added in the basic cryopreserving liquid of 10ml, be mixed with the mother liquor of 105ng/ml;

Get again mother liquor 1 μ l and be diluted in the above-mentioned basic cryopreserving liquid of 10ml, obtain the cryopreserving liquid that required final concentration is 10ng/ml.

Cell cryopreservation:

Cell is through 1000rpm, and the centrifugal supernatant of outwelling of 10min is resuspended with the cryopreserving liquid containing barren wort total chromocor, packs in aseptic cell cryopreservation tube.

Frozen program:

Put into programmed cell freeze box, after-80 ℃ of placement 12h, put into liquid nitrogen and preserve for a long time.

Under 4 ℃ of conditions, place 30min, place 2h for-20 ℃, after-80 ℃ of placement 16-18h, directly put into liquid nitrogen and preserve for a long time.

Cell recovery:

Cryopreservation tube, from liquid nitrogen takes out, stirs to dissolving rapidly in 37～40 ℃ of waters, adds DMEM medium, and the centrifugal 10min of 1500rp, abandons supernatant, puts in 37 ℃, 5%CO2 incubator and cultivates.

Platform is expected blue dyeing counting cytoactive rate (in Table 1).

Activity ratio after the recovery of table 1. mesenchymal stem cells MSCs

Claims (2)

1. one kind for the long-term cryopreserving liquid of preserving adult stem cell, it is characterized in that containing in the cryopreserving liquid of every 100ml basis: barren wort total chromocor 10～20ng/ml, and in the cryopreserving liquid of every 100ml basis, contain: DMEM medium 50%～70%, dimethyl sulfoxide (DMSO) 10%, Knock-out serum 40%～20%.

Claim 1 for the long-term preparation method who preserves the cryopreserving liquid of adult stem cell, comprise the following steps:

In dimethyl sulfoxide (DMSO): Knock-out serum: DMEM medium ratio is that 1:2:7 concentration is prepared basic cryopreserving liquid;

1mg barren wort total chromocor is added in the DMEM culture fluid of 10ml, be mixed with the mother liquor of 105ng/ml, then get mother liquor 1 μ l and be diluted in the above-mentioned basic cryopreserving liquid of 10ml, obtain the cryopreserving liquid that required final concentration is 10ng/ml.