Porcine bone marrow mesenchymal stem cells

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Product Name:Porcine bone marrow mesenchymal stem cells

Organizational source:bone marrow

Product Specifications5×105cells/T25cell culture flask

Training Information:

The culture medium containsFBSGrowth additivesPenicillinTheStreptomycinwait

Fluid change frequency per2-3Change the fluid once every day

Growth characteristics: Wall adhesion

Cell morphology fibroblast like

Passage characteristics can be passed down5Dai Zuo;3Excellent condition within the generation

digestive juice0.25%

Cultivation conditions gas phase: air,95%；CO2，5%

Cell Introduction:

Porcine bone marrow mesenchymal stem（BMSC）Separated from bone marrow; Bone marrow is the hematopoietic tissue of the body, located within many bones. The bone marrow of adult animals is divided into two types: red bone marrow and yellow bone marrow. Red bone marrow can produce red blood cells, platelets, and various white blood cells. Platelets have hemostatic effects, while white blood cells can kill and inhibit various pathogens, including bacteria, viruses, etc; Some lymphocytes can produce antibodies. Therefore, the bone marrow is not only a hematopoietic organ, but also an important immune organ. Bone marrow is a spongy tissue that exists between the medullary cavity of long bones (such as humerus and femur) and the trabecular bone of flat bones (such as ilium). The bone marrow that can produce blood cells is slightly red in color and is called red bone marrow. At birth, the red bone marrow fills the entire bone marrow cavity. As age increases, the number of fat cells increases, and a considerable portion of the red bone marrow is replaced by yellow bone marrow. Finally, almost only the flat bone marrow cavity contains red bone marrow. The bone marrow mesenchymal stem cells present in the bone marrow stromal system are a type of stem cell with high self-renewal ability and multi-directional differentiation potential, in addition to hematopoietic stem cells. They can differentiate into various tissues such as bone, cartilage, muscle tissue, skin, fat, and nerves, and can therefore serve as seed cells in tissue engineering. In the bone marrow,BMSCProportion of total nucleated cells in bone marrow0.001%-0.1%The content is extremely low. The in vitro culture conditions for bone marrow mesenchymal stem cells require high requirements. During the culture process, factors such as adhesion time, implantation density, serum content, culture temperature, and culture medium are importantpHThe influence of conditions such as values.

Method Introduction:

The pig bone marrow mesenchymal stem isolated in the company's laboratory was prepared by washing the bone marrow, density gradient centrifugation, differential adhesion method combined with culture medium screening. The total number of cells was approximately5×10?cells/Bottle.

Quality inspection:

Porcine bone marrow mesenchymal stem cells isolated in the company laboratoryCD90Immunofluorescence identification, purity can reach90%Above, and not containingHIV-1TheHBVTheHCVMycoplasma, bacteria, yeast, fungi, etc.

Cultivation steps:

1、 Resuscitate cells: Quickly shake and thaw a cryovial containing 1mL of cell suspension in a 37 ℃ water bath, then add 5mL of culture medium and mix well. Centrifuge at 1000 RPM for 5 minutes, discard the supernatant, add 4-6 mL of culture medium, and blow evenly. Then add all cell suspensions to the culture bottle and culture overnight (or add the cell suspensions to a 6cm dish), and culture overnight. The next day, change the solution and check the cell density.

2、 Cell passage: If the cell density reaches 80% -90%, passage culture can be carried out.

a）、 For adherent cells, the following methods can be used for passaging:

1. Discard the culture supernatant and rinse the cells 1-2 times with PBS that does not contain calcium or magnesium ions.

2. Add 1-2ml of digestion solution (0.25% Trypsin 0.53mM EDTA) to a culture bottle, place it in a 37 ℃ incubator for digestion for 1-2 minutes, and then observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take them back to the operating table, lightly tap the culture bottle a few times, and add 5ml or more of culture medium containing 10% serum to terminate digestion.

3. Gently blow the cells, remove and aspirate, centrifuge at 1000RPM for 8-10 minutes, discard the supernatant, add 1-2mL of culture medium, and blow evenly.

4. Add 5-6ml/bottle of culture medium and divide the cell suspension into new dishes or bottles containing 5-6 ml of culture medium in a ratio of 1:2.

b）、 For suspended cells, the following methods can be used for passaging:

Method 1: Collect cells, centrifuge at 1000RPM for 8-10 minutes, discard the supernatant, add 1-2ml of culture medium and blow evenly. Divide the cell suspension into new dishes or bottles containing 8ml of culture medium in a ratio of 1:2 to 1:5.

Method 2: You can choose the method of half changing the medium. After discarding half of the medium, suspend the remaining cells and divide the cell suspension into new dishes or bottles containing 8ml of medium in a ratio of 1:2 to 1:3.

How to handle received cells?

1. Firstly, observe whether the cell culture bottle is intact and whether there is any leakage or turbidity in the culture medium. If there are any, please take photos and contact technical support in a timely manner (the photos taken will be used as the basis for subsequent services).

2. Wipe the surface of the cell culture bottle with 75% alcohol and observe the cell status under a microscope. Due to transportation issues, a small amount of adherent cells may detach from the bottle wall; Do not open the culture bottle cap yet, place the cells in the cell culture box and let them stand for 2-4 hours to stabilize the cell state.

3. Carefully read the cell manual to understand cell related information, such as adhesion characteristics (adhesion/suspension), cell morphology, basic culture medium used, serum ratio, required cytokines, passage ratio, fluid exchange frequency, etc.

4. After settling, take out the cell culture bottle, examine it under a microscope, take photos, and record the cell status (the photos taken will be used as the basis for subsequent services); It is recommended to regularly take photos and record the growth status of cells after subculture.

5. Adherent cells: If the cell growth density exceeds 80%, it can be passaged normally; If the cell density does not exceed 80%, remove the culture medium from the cell culture bottle and reserve about 5ml for further cultivation until the cell density reaches around 80% before proceeding with the passage operation. The bottle cap can be slightly loosened.

6. Suspended cells: Transfer all the liquid in the cell culture bottle to a 50ml sterile centrifuge tube and centrifuge at 1200rpm for 5 minutes. After centrifugation, collect the supernatant culture medium for later use. Add 5ml of culture medium to the bottom of the tube and resuspend the cell precipitate. During microscopic examination, if the cell density exceeds 80%, the cell suspension can be cultured in two cell culture bottles and supplemented with culture medium up to 5ml. If the cell density does not exceed 80%, the cell suspension can be transferred to the original bottle for further cultivation until the cell density reaches around 80% before passaging

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