A cryopreservation solution for human spermatogonial stem cells

Abstract

The invention relates to cryopreservation liquid of human spermatogonial stem cells. Specifically, the cryopreservation liquid of the human spermatogonial stem cells, provided by the invention, is prepared from a basic culture solution and an activity-keeping peptide. The cryopreservation liquid has the advantages of simple components, convenience for preparation and low cost; disordered differentiation of the human spermatogonial stem cells is not caused by special stimulating substances; especially, the activity-keeping peptide is added and the cryopreservation safety and stability of the human spermatogonial stem cells can be greatly improved, so that the cell viability is improved and the cryopreservation effect is remarkably better than that of common cell cryopreservation liquid; thecryopreservation liquid can be used for long-period preservation and application of the human spermatogonial stem cells.

Description

technical field

[0001] The invention relates to the technical field of stem cells, in particular to a cryopreservation solution of human spermatogonial stem cells. Background technique

[0002] Spermatogonia stem cells (SSCs) refer to a type of primitive spermatogonia located on the basement membrane of the seminiferous tubules, which can not only self-renew to maintain a constant amount of itself, but also differentiate into spermatocytes. With the birth of male animals, primordial germ cells transform into gonocytes, migrate to the basement membrane of the seminiferous tubules, and differentiate into spermatogonial stem cells.

[0003] In vivo, spermatogonial stem cells (SSCs) are a type of primordial spermatogonia located on the basement membrane of the seminiferous tubules, which can maintain their own number through self-renewal, differentiate into spermatocytes, and further form mature sperm. In 1994, Brinster et al verified the existence of SSCs through germ cell tra...

Examples

Embodiment 1

[0019] Embodiment 1, preparation of human spermatogonial stem cells

[0020] The testicular tissues of male fetuses legally induced labor were digested with type Ⅰ collagenase and trypsin successively, followed by Percoll density gradient centrifugation and differential adherence method, to isolate and culture human spermatogonial stem cells, and to carry out the isolation and culture of human spermatogonial stem cells by cell immunofluorescence method. Identification; Flow cytometry immunofluorescence method is used to screen Oct-4 marker-positive cells, and finally obtain spermatogonial stem cells (such as figure 1 shown). Of course, other methods of obtaining spermatogonial stem cells in the field can also be used, which is a conventional choice in the prior art.

Embodiment 2

[0021] Preparation of Example 2 Cell Freezing Liquid

[0022] Artificially synthesize the active peptide shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0023] The configuration of the basic culture medium: add 1.0% sucrose, 400.5% dextran, 0.2% glutathione, 0.6% glutamine, 0.5% trehalose, and L-glutamine to 1640 sterile medium 0.1%, glycerol 15% (V / V), use 1640 sterile medium to make up to 1L.

[0024] The added amount of the activity maintaining peptide in the basal culture solution is 0.3%.

[0025] Preparation of control freezing solution 1: 5 mg / ml low-density lipoprotein and 0.5 mg / ml lecithin are compatible with antifreeze.

[0026] Preparation of control freezing solution 2: Cell membrane protective agent 1%: composed of sucrose 0.5%, polymer anhydride-5000.5%, DMSO 8%; cell sedimentation stabilizer 25%: composed of methylcellulose 4000CP 15% , hydroxyethyl starch 10%; antioxidant O.2%: composed of vitamin C O.1%, glutathione O.1%; cell nutrition 1.0%, composed of glutamin...

Embodiment 3

[0027] Example 3 Cryopreservation of human spermatogonial stem cells 1

[0028] The human spermatogonial stem cells prepared in Example 1 were cultured in 1640 sterile medium and 0.3% of the active peptide of SEQ ID NO: 1, at 37°C, 5% CO 2 Cultured under the conditions of 2 days (polypeptides can stimulate cells to produce antifreeze substances), collect cells, culture in the basal culture solution of Example 2+SEQ ID NO: 1 active peptide 0.3%, at 30 ℃, 5% CO 2 Cultivate for 12 hours under certain conditions, and store directly in liquid nitrogen.

technical field

[0001] The invention relates to the technical field of stem cells, in particular to a cryopreservation solution of human spermatogonial stem cells. Background technique

[0002] Spermatogonia stem cells (SSCs) refer to a type of primitive spermatogonia located on the basement membrane of the seminiferous tubules, which can not only self-renew to maintain a constant amount of itself, but also differentiate into spermatocytes. With the birth of male animals, primordial germ cells transform into gonocytes, migrate to the basement membrane of the seminiferous tubules, and differentiate into spermatogonial stem cells.

[0003] In vivo, spermatogonial stem cells (SSCs) are a type of primordial spermatogonia located on the basement membrane of the seminiferous tubules, which can maintain their own number through self-renewal, differentiate into spermatocytes, and further form mature sperm. In 1994, Brinster et al verified the existence of SSCs through germ cell tra...

Examples

Embodiment 1

[0019] Embodiment 1, preparation of human spermatogonial stem cells

[0020] The testicular tissues of male fetuses legally induced labor were digested with type Ⅰ collagenase and trypsin successively, followed by Percoll density gradient centrifugation and differential adherence method, to isolate and culture human spermatogonial stem cells, and to carry out the isolation and culture of human spermatogonial stem cells by cell immunofluorescence method. Identification; Flow cytometry immunofluorescence method is used to screen Oct-4 marker-positive cells, and finally obtain spermatogonial stem cells (such as figure 1 shown). Of course, other methods of obtaining spermatogonial stem cells in the field can also be used, which is a conventional choice in the prior art.

Embodiment 2

[0021] Preparation of Example 2 Cell Freezing Liquid

[0022] Artificially synthesize the active peptide shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0023] The configuration of the basic culture medium: add 1.0% sucrose, 400.5% dextran, 0.2% glutathione, 0.6% glutamine, 0.5% trehalose, and L-glutamine to 1640 sterile medium 0.1%, glycerol 15% (V / V), use 1640 sterile medium to make up to 1L.

[0024] The added amount of the activity maintaining peptide in the basal culture solution is 0.3%.

[0025] Preparation of control freezing solution 1: 5 mg / ml low-density lipoprotein and 0.5 mg / ml lecithin are compatible with antifreeze.

[0026] Preparation of control freezing solution 2: Cell membrane protective agent 1%: composed of sucrose 0.5%, polymer anhydride-5000.5%, DMSO 8%; cell sedimentation stabilizer 25%: composed of methylcellulose 4000CP 15% , hydroxyethyl starch 10%; antioxidant O.2%: composed of vitamin C O.1%, glutathione O.1%; cell nutrition 1.0%, composed of glutamin...

Embodiment 3

[0027] Example 3 Cryopreservation of human spermatogonial stem cells 1

[0028] The human spermatogonial stem cells prepared in Example 1 were cultured in 1640 sterile medium and 0.3% of the active peptide of SEQ ID NO: 1, at 37°C, 5% CO 2 Cultured under the conditions of 2 days (polypeptides can stimulate cells to produce antifreeze substances), collect cells, culture in the basal culture solution of Example 2+SEQ ID NO: 1 active peptide 0.3%, at 30 ℃, 5% CO 2 Cultivate for 12 hours under certain conditions, and store directly in liquid nitrogen.

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