Basic techniques for mammalian cell tissue culture tritech. This allows the synthesis of a tissue equivalent or construct on demand for basic studies on cellcell and cellmatrix interaction and for in vivo implantation. Cell culture cell culture is one of the major tools used in cellular and molecular biology, providing excellent model systems for studying the normal physiology and biochemistry of cells e. For these types of cell lines subculture by dilution is relatively easy. Tip ensure the neurons are evenly suspended before removing an aliquot. The handbook is intended as a guide rather than an indepth text book of cell culture and you are encouraged to consult relevant specialised literature to. Cell culture techniques are ubiquitous in areas of developmental biology, drug discovery, regenerative medicine and protein production. Introduction primary culture of hepatocytes is an in vitro model widely used to investigate. Introduction primary culture of hepatocytes is an in vitro model widely used to investigate various aspects of liver physiology and pathology 1. Bring cell media, pbs, and trypsin to room temperature. Dislodge cells from the flask substrate with a cell scraper. Cell cloning by serial dilution in 96 well plates protocol introduction this technique is widely used for clonal isolation of hybridomas and other cell lines that are not attachment dependent. For example, put the required volumes of cell media into the new dishes. Wicell recommends that stem cells should be thawed and established in the conditions in which they were initially frozen prior to transfer to alternate culture platforms.
Procedure 1 sanitize the cabinet using 70% ethanol before commencing work. Overall, an aspect of pharmaceutical research which promisingly employs cell culture models is the study of in vitro drug transportabsorption and metabolism. However, the precise category required is dependent upon the cell line and nature of the work proposed. Cell culture protocols thermo fisher scientific us. We therefore recommend that you familiarize yourself with your cell line of interest, and closely follow the instructions provided with each product you are using in your experiments. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete.
General protocol for recovering or freezing primary cells. In fixed cells, the mycofluor reagent has access to the cell. Animal cell culture protocol aseptic technique and good cell culture practice to ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. Viral growth medium is usually supplemented with a lower percentage of serum than cell growth medium, often ranging. If plating hepatocytes with an overlay, refer to the specification section for matrigel coating which will provide the protocol and technical tips. Transfer vero cell suspension to tissue culture flask with vented cap. Mammalian cell tissue culture techniques protocol abcam.
The cultures were tested using the invitrogen mycofluor mycoplasma. Although cell culture can theoretically be carried out on an open bench in a lowtraffic area, most cell culture work is carried out using a horizontal laminarflow clean bench see basic protocol 2 or a vertical laminarflow. Any and all media, supplements, and reagents must be sterilized by filtration through a 0. Resuspend cell pellet with the complete and dispense into the culture flasks from step 1. Heterogeneous primary culture isolate by cloning, selective media, andor cell sorting perfused multilayer from monolayer sponge or scaffold perfused capillary bed spheroid or organoid filter well insert propagate and seed on desired substrate. The following protocol is for passaging hela cells that are. Nowadays, animal cell culture becomes a reasonable alternative for animal experiments in the process of drug discovery and development. The viability of cell banks is dependent on the cryopreservation procedure employed when making them and on the proper storage conditions. Gibco cell culture heroes meet the researchers driving tomorrows breakthroughs in the fight against cancer. However, it is also very useful for cloning attachment dependent cells when the cell plating efficiency is very low, unknown or unpredictable. Dispense a 5 l droplet of the cell suspension 140,000 neurons directly over the recording electrode area of each well of the. Resuspend the cell pellet in 10 gml laminin solution to a final concentration of 28,000 viable neuronsl. When subculturing cells 6075% confluency, discard old media from flask. The basic protocol describes subculturing of a monolayer culture grown in.
Troubleshooting tips problem observation of problem possible causes and solutions spontaneous differentiation within culture image 2 differentiation can look different depending on culture and cause of. The following is a general guideline for culturing of cell lines. The cells can be of a mixed, heterogeneous origin with different cell types growing, or they can be a singular cell type, sometimes clonal in origin. All cell culture must be undertaken in microbiological safety cabinet using aseptic technique to ensure sterility. Protocols specific to individual tissue types will be presented in. Human embryonic stem cell culture basement membranes are continuous sheets of specialized extracellular matrix that are found at the dermalepidermal junction, at the base of all lumenlining epithelia throughout the digestive, respiratory, reproductive, and urinary tracts, and that underlie parenchyma of endocrine and exocrine glands.
Cell culture is a method used to cultivate, propagate and grow a large amount of cells in a dish. Transfer the vial contents to a centrifuge tube containing 9. Remove vial cap using little finger of same hand that holds inoculating loop. Immunocytochemistry and immunofluorescence protocol.
Boldly determined and deeply committed, our cell culture heroes work tirelessly to lay the foundation for discoveries that may lead to cures. Cell lines are routinely frozen to make and keep referenceparental cell lines, newly produced transgenic cell lines, keep stocks of primary and immortalized cells, and for shipping purposes. To that aim, we have assembled this updated laboratory handbook of cell culture techniques. Cell culture guidelines the following is a general guideline for culturing of cell lines.
Bacterial culture techniques 337 if working from stab culture. Since the introduction of cell culture techniques, cells have been cultured in twodimensions, attached to tissue culture plasticware or ecm attachment pro. Discard the cell culture medium by inverting the slide and gently tapping it on a paper towel to remove the remaining medium. Hood regulations a close hood sash to proper position to maintain laminar air flow b avoid. Special techniques cell pellet protocol this protocol is modified from joseph garaots, m. Cell culture on microelectrode arrays axion biosystems. Jurkat atcc number tib152 cell culture and formaldehyde. However, some cells, particularly primary cells, will require growth on special. The cell provider does not recommend centrifugation and is not responsible for cell death induced by centrifugation. Current protocols in cell biology wiley online library. Norma neff and tim reddy jurkat atcc number tib152 cell culture and formaldehyde crosslinking jurkat clone e61 is a human t lymphoblastoid cell line derived from an acute t cell leukemia. Fundamental techniques in cell culture sigmaaldrich. Cell culture protocols thermo fisher scientific tw. The consequences of deviating from the culture conditions required for a particular cell type can range from the expression of aberrant phenotypes to a complete failure of the cell culture.
This is not only meant to prevent the contamination of the cells, but to also ensure that the researchers themselves are protected from any form of contamination. In advance, prepare the cell growth medium for growing the host cell line. Breseagen protocol human embryonic stem cell protocols. Cell culture protocol cell culture protocols are meant to ensure that culture procedures are carried out to the required standards. The cultures were tested using the mycofluor mycoplasma detection kit, following the kit protocols. The handbook is intended as a guide rather than an indepth text book of cell culture and you are encouraged to consult relevant specialised literature to obtain more detailed information. When cells reach a 90% confluent monolayer, passage cells into new tissue culture flasks see basic protocol 2. A procedure to concentrate cells from suspension culture or to resuspend cells from a monolayer culture. Use aseptic technique to prevent microbial contamination. The protocol below describes how to subculture adherent and suspension cultured cells. All cell culture procedures must be conducted in a biosafety cabinet. Quickly pass mouth of vial several times through burner flame. How to count and calculate the number of cells from a stock flask or culture dish.
Mouse embryonic stem cell culturing protocols form 105 rev b072214 5 of 6 table 2. Breseagen protocol human embryonic stem cell protocols this material was cultured and frozen using bresagens protocols. Prepare the new dishes andor six well plates which will be used for the new split. Fix the cells with 4% formaldehyde diluted in 1x pbs prepare fresh for 10 min at room temperature fixation time can be increased to 20 min depending on the cell line. A more comprehensive reference on animal cell culture. For the researcher new to cell culture, this handbook. Note that cell culture conditions vary for each cell type. Cell culture is the process by which prokaryotic, eukaryotic or plant cellsare grown under controlled conditions in practice it refers to the culturing of cells derived from animal cells. This section provides guidelines and general procedures for routine subculturing, thawing, and freezing of cells in culture.
This can be performed either simultaneously in a mixture or sequentially one antigen after another. Cell culture was first successfully undertaken by ross harrison in 1907 roux in 1885 for the first time maintained. Coat coverslips with polyethylineimine or polyllysine for 1 h at room temperature. This protocol assumes that the cells are being cultured in t150 flasks equipment equipment. Cell culture protocol fcdi icell glutaneurons neurons are sensitive to centrifugation, so care should be taken to monitor speed and duration during this step. Icc and if protocol multicolor immunostaining optional step to examine the codistribution of two or more different antigens in the same sample, use a double immunofluorescence procedure.
1543 992 1101 999 1520 980 57 193 57 1522 1141 269 407 1431 75 171 1344 64 104 122 1308 848 1535 223 454 1176 408 1240 77 368 86 1474 933 844 461