Establish a cell line or cell line Various cell lines or cell lines that have been named and identified by cell biology are all cell groups with relatively uniform morphology, stable growth and proliferation, and clear biological characteristics. Therefore, any cell group that meets the above conditions can also be given a corresponding name, which is often referred to in the literature as identified cells (Certified Cells). The identified cells can be used in various experimental studies and production of biological products. The various cell lines (strains) that have been established in the world have been innumerable, and more than one hundred have been established in China, and they are constantly growing.
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1. Types and names of cells cultured in vitro The names of cells cultured in vitro evolve with the development of cell culture technology and the increase of cell types. The earliest name used was cell strain, and the term "cell line" appeared in the future. The two were used together for a time, and the concept was unclear, which caused confusion in the literature. China has had a similar situation. Before our country has formulated a unified noun, the nouns used in this book basically refer to Schaeffer, WI (1979) and related domestic conferences, and commonly used nouns in domestic and foreign magazines.
(1) Primary culture Primary culture, also known as primary culture, is the first culture of cells, tissues and organs taken directly from the body. Once the cells have been subcultured (Subculture), they are no longer called primary cultures, but are instead called cell lines.
(2) The cell after the primary culture of the cell line has been cultured for the first time. If the cell line has a limited survival period, it is called a finite cell line (Finite Cell Line); a cell line that has been able to survive continuously with unlimited reproduction capacity is called a continuous cell line or an infinite cell line (Infinite Cell Line). Most of the infinite cell lines have undergone aneuploidy, with aneuploid karyotype, and some may have become malignant cells, so they are essentially transformed cell lines. Some of the infinite cell lines are only immortal (or immortal), but still retain contact inhibition and no carcinogenicity of allogeneic vaccination; some are not only immortal, but allogeneic vaccination is also tumorigenic, indicating that they have been malignant. These two infinite cell lines with different properties are often not very strict in the application of these terms in domestic and foreign literature. For clarity of concept, it may be more appropriate to use the term "malignant transformed cell line" for malignant infinite cell lines in this book. For those cell lines that are only immortal and not malignant, infinite cell lines or transformed cell lines can be used. The currently circulating NIH3T3, Rat-1, 10T1 / 2, etc. belong to this type of cell line.
A cell line isolated from a cell line that differs from the original cell line in traits is called a subline of that cell line (Subline).
(3) The cloned cell line is isolated from a biologically identified cell line by single cell isolation or culture, or by screening. The cell group formed by single cell proliferation is called a cell line. Then, the original cell strain is further used to isolate and culture a cell group with different characteristics from the original strain, which can also be called a substrain (Substrain)
(4) The chromosome number of the diploid cell cell group has the same or substantially the same chromosome number as the original donor diploid cell (2n cells account for 75% or 80% or more), which is called diploid cell culture. If only the number is the same, and the karyotype is different, that is, the chromosome morphology has changed is pseudodiploid. Diploid cells have a limited life span under normal circumstances and are therefore limited cell lines. However, the life span of diploid cells varies with the age of donors and tissue cells. Human embryonic lung fibroblasts can be transmitted for 50 generations and 10 generations, human embryonic kidneys only have 8 to 10 generations, and human embryo glial cells are 15 to 30 generations; if they can be transferred from elderly individuals, the cell survival period is shorter. Diploid cell lines established from donors of different ages can be used to study aging. In order to keep diploid cells to be used for a long period of time, they are generally stored in the first generation or 2 to 5 generations as a stock (Stook Cells), and then propagated when they are used, and then kept frozen after use. Aging.
(5) Genetically deficient cells: Cells cultured from those with congenital genetic defects (mainly fibroblasts), or cells that have induced mutations manually are genetically deficient cells. Such cells may have a diploid karyotype or aneuploid.
(6) Tumor cell lines or strains This is the most common type of existing cell lines. The established cell lines in China are mainly such cells. Tumor cell lines are mostly built from carcinomas, mostly epithelial-like cells, often passed through dozens of generations or more than a hundred generations, and are non-destructive and tumorigenic with allogeneic vaccination.
It is customary to give names to various cell lines or cell lines that have been established; there is no strict unified regulation for the naming of cells, and most of them are expressed by abbreviations or codes with a certain meaning. However, no matter what form, it should not be too long for easy memory and understanding. I will not cite the following representative cell names for reference:
HeLa: the name of the donor patient (from cervical cancer)
CHO: Chinese Hamster Ovary
Gong-743: cervical cancer epithelial cells, established in March 1974
NIH3T3: Established by the National Institute of Health; subcultured every 3 days, each time inoculated with 3 × 105 cells / ml.
2. Requirements for the establishment of cell lines (or strains) Regarding what kind of in vitro cultured cell populations can be confirmed as certified cells, there are no uniform regulations in the world, generally depending on the specific situation . When used only as primary cultured cells, as long as the donor's sex, age, etc. are uniform, the conditions of the material source and tissue type are stable, there are not many items for identification, and there are several items that can explain the relevant traits of the cells. If it can be stored for a long time and can be used by other research laboratories, especially for cells that are repeatedly passaged, it is customary to have the following requirements, and it should be explained when reporting in the journal.
(-) The source of the tissue should indicate the species to which the cell donor belongs, from the human body, animal or other; the donor's age, sex, organ or tissue taken from; if it is a tumor tissue, the clinical pathological diagnosis, tissue source, and case number should be stated Wait.
(2) Cell biology testing should understand the general and special biological traits of the cell, such as the general morphology, specific structure, cell growth curve and division index, doubling time, inoculation rate of the cell; specificity, such as whether there are special products for gland cells Including secreted proteins or hormones; if it is a tumor cell, try to prove that the cell is indeed derived from the original tumor tissue and not other, and still maintain sexuality. For this purpose, soft agar culture, allogeneic animal inoculation and tumorigenicity are required. Experiments such as tissue infiltration.
(3) Culture conditions and methods Various cells have their own relatively suitable living environment. Therefore, the medium used, the type of serum, the dosage, and the appropriate pH for cell survival should be specified.
3. The identification, management and use of established cell lines or strains In recent years, when a cell line or cell forest is completed, China often conducts identification by organizing expert identification meetings, which is not necessary from an academic point of view. . According to international practice, as long as the research reports the relevant materials in magazines or journals and introduces the above items in detail.
In the past, when a unified cell bank was not established, most of the established cell lines (strains) were kept by the authors themselves, which was a waste of manpower, inconvenience in communication and use, and easy contamination and loss of cells. China has initially established a small-scale cell storage mechanism, which needs to be further developed, which will certainly have a greater role in promoting cell culture in China.
Internationally, the United States, Britain and Japan have established cell banks; the United States has the American Standard Cell Bank or Cell Bank (ATCC), Human Genetic Mutation Cell Bank (HGMR), and Cell Senescence Cell Bank (CAR), among others.
ATCC is not only the largest cell bank in the United States but also in the world. ATCC has a group of collaborative laboratories and an advisory committee composed of many experts. ATCC is also a resource bank for the National Cancer Institute (NCI) and the National Institutes of Health (NIH), and has a close relationship with NCI. ATCC is also the International Culture Cell Literature Center of the World Health Organization WHO. ATCC currently stores 3200 cell lines that have been identified in liquid nitrogen (1992), including skin fibroblast cell lines from normal people and patients with various diseases; and nearly 75 hybridoma cell lines from different species. ATCC accepts cells from countries around the world that have been identified for storage, and also provides free research cells to researchers or laboratories around the world (charges are made for profitable research.) When ATCCs are included in pooled cells, they must meet their storage standards The test items required for ATCC storage cells are as follows:
Cultivation resume: tissue source date, species, tissue origin, gender, age, donor normal or abnormal health status, cell passage number, etc. Cryopreservation medium: culture medium and antifreeze name Cell viability: cell inoculation survival rate and growth before and after thawing Characteristic culture medium: medium type and name (generally requires no antibiotics), serum source and content Cell morphology: type, such as epithelial or fibroblast cells, etc. Cell growth characteristics after thawing Karyotype: diploid or polyploid , Detection of the presence or absence of contamination of marker chromosomes: including bacteria, fungi, mycoplasma, protozoa and viruses and other species: detection of isozymes, mainly G6PD and LDH, to prove whether cells are cross-contaminated and reverse transcriptase detection immunoassay : One or two serological test cell creators: the name of the founder; the name of the detector above is the basic requirements for ATCC storage, and the hybridoma storage standards are still different.