Establishment of cell lines or cell lines

1. Concept
1. Cell Line: The primary culture Wujing is the first time to successfully pass into a cell line. It consists of the lineage of cells that originally existed in the primary culture.
2. Cell strain (Cell Strain): The cell strain is obtained from the primary culture or cell line by the selection method or the cloning method with special properties or markers. The special properties or signs of cell lines must be present throughout the culture period. If it is not possible to continue passage or the number of passages is limited, it is called a finite cell strain; if it can be passaged continuously, it is called a continuous cell strain (continuous cell strain).
strain). For human tumor cells, those that have been cultured in vitro for more than half a year, grow stably, and are serially passaged are called continuous strains or lines.

2. Requirements for the establishment of cell lines (or strains) What kind of in vitro culture group can be recognized as the identified cells depends on the specific situation and there is no uniform regulation. As for the cells used for primary culture, as long as the donor is uniform and the conditions such as the location of the material and the type of tissue are stable, if it is used for long-term culture, especially cells that are passaged repeatedly, the following instructions are often required:
1. Tissue source: the species to which the cell donor belongs, from humans or animals;
The individual's gender and age; the organ or tissue from which the material was obtained.
If it is a tumor tissue, the clinical and pathological diagnosis, and the medical record number should be stated.
2. Cell biology test:
Understand the general and special biological traits of cells, such as general cell morphology, specific structure, cell growth curve and division index, doubling time, inoculation rate, etc.
If it is a tumor cell, in order to explain that it is derived from the original tumor tissue and remains malignant, it must be subjected to soft agar culture, tumor allogeneic inoculation and invasion of normal tissues.
3. Culture conditions and methods; the living environment adapted to the cell line (or strain) should be stated, that is, the medium used, the type of serum, the dosage and the appropriate PH value should be indicated.

3. The main points and basic process of cell construction

(1) Technical points of tumor cell culture
1. Material: The material mainly comes from surgical operation or biopsy of tumor tissue. Avoid using necrotic tissue when selecting materials. Choose the areas with concentrated and viable tumor cells. Tumor metastasis lymph nodes or pleural and ascites are better culture materials. Cultivate as soon as possible after taking the material. Those who cannot be immediately cultivated for some reason can be stored frozen. The cultivation method and freezing method are the same as the normal tissues mentioned above.
2. Fibroblast exclusion: Some fibroblasts are often mixed in the tumor tissue, which can grow simultaneously with the tumor cells during culture, and often pressurized the cancer cells, causing the growth of cancer cells to be blocked or even disappear, and should be carefully excluded.
Exclusion methods often include: mechanical scraping method, repeated adherence method, digestion elimination method, collagenase digestion method, etc.
3. Improve the survival rate and growth rate of tumor cell culture According to experimental experience, tumor cells are not easy to culture in vitro, and it is more difficult to establish a tumor cell line that can be passaged. Generally, when the tumor composition or cells are primary cultured, they must grow to adapt to the new environment. Therefore, they cannot be limited to general culture methods, and some special measures must be adopted. For example: use suitable substrate, bottom layer of rat tail collagen and bottom layer of feeder cells. With cell growth factors, different cell growth factors such as insulin, hydrocortisone, estrogen, etc. are selected according to the type of cells. Animal culture can also be considered.

(2) Method of establishing human lymphoblasts
1. Put 4ml of heparin anticoagulated blood into the centrifuge tube and add 2ml of RPMI 1640.
2. After mixing, slowly add along the wall of the tube to the liquid surface preset with 4ml of lymphocyte separation solution and let stand for 30min.
3. At 1500 rpm for 15 minutes, draw the leukocyte layer (second layer) into another centrifuge tube.
4. Add 5ml of RPMI 1640 to wash the white blood cells twice.
5. Inoculate leukocytes into 1ml RPMI 1640 medium, add 10μl cyclosporine and 100μl EBV (EBV) solution, and mix well.
6. Water bath shaker, 40 times / min, 37 ℃, 3hr.
7. 1500rpm, 15min. Inoculate the cells into 1ml medium (containing glutamine 1mM / ml), add 10μl cyclosporine, mix gently and incubate at 37 ℃.
8. Observe the cell transformation and growth after 5 days, and decide whether to change the medium in half. Generally, change the fluid in half a volume 1-2 times, and maintain the concentration of cyclosporine.
9. After the number of cells to be transformed has risen significantly, and there are cell clumps, it can be transferred to a 25ml cell culture flask, add 1-2ml of culture medium, and cultivate at 37 ℃ for 10-15 days, generally observe every 3-4 days Decide whether to change fluid and pass it on.
10. After the cells have grown to a certain amount, they should be frozen and stored. Karyotype analysis and archiving should be carried out before freezing.

A diaper (American and Canadian English) or a nappy (Australian and British English) is a type of underwear that allows the wearer to defecate or urinate without the use of a toilet, by absorbing or containing waste products to prevent soiling of outer clothing or the external environment. When Diapers become wet or soiled, they require changing, generally by a second person such as a parent or caregiver. Failure to change a diaper on a sufficiently regular basis can result in skin problems around the area covered by the diaper.

Diapers are made of cloth or synthetic disposable materials. Cloth diapers are composed of layers of fabric such as cotton, hemp, bamboo, microfiber, or even plastic fibers such as PLA or PU, and can be washed and reused multiple times. Disposable Diapers contain absorbent chemicals and are thrown away after use.

Diapers are primarily worn by infants, toddlers who are not yet potty trained, and by children who experience bedwetting. They are also used by adults with incontinence, in certain circumstances where access to a toilet is unavailable or for psychological reasons. These can include those of advanced age, patients bed-bound in a hospital, individuals with certain types of physical or mental disability, diaper fetishists, and people working in extreme conditions, such as astronauts. It is not uncommon for people to wear diapers under dry suits.

Babies may have their diapers changed five or more times a day. Parents and other primary child care givers often carry spare diapers and necessities for diaper changing in a specialized diaper bag. Diapering may possibly serve as a good bonding experience for parent and child. 

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