Primary Culture Chapter 12

What is Primary Cell Culture? That stage of culture obtained after isolation of cells but before first subculture

How can you obtain Primary Cell Culture? Stages: 1. Acquisition of sample 2. Isolation of tissue 3. Dissection and/or disaggregation 4. Culture after seeding into culture vessel After isolation a PCC can be obtained either by allowing cells to migrate out from fragments of tissue adhering to a suitable substrate or by disaggregating the tissue mechanically or enzymatically to produce a suspension of cells that can attach to a substrate

What are the different enzymes used for tissue disaggregation? Trypsin, Collagenase, Elastase, Pronase, Dispase, DNAase and Hyaluronidase or in various combinations (Mammalian enzymes) Trypzean, TrypLE, Accutase and Accumax (Nonmammalian enzymes) Trypsin is the common enzyme used

Effectiveness of different enzymes Trypsin and Pronase give complete disaggregation Collagenase and dispase give incomplete disaggregation Trypsin and Pronase may damage the cells Collagenase and dispase are less harmful Show effects on cell viability and yield. Trypsin is a pancreatic protease with specificity for peptide bonds involving the carboxyl group of basic amino acids arginine and lysine. It is obtained from pancreas of porcine Hyaluronidase + Collagenase digests intracellular matrix DNase – disperses DNA released from lysed cells

What are the set of conditions required commonly by most of the cultures? Fat and necrotic tissue are removed during dissection Chopping should be fine with sharp instruments Enzymes used for disaggregation should be removed Concentration of cells – primary > subculture Choice of Rich medium Choice of Embryonic tissue Chopping should lead to less tissue damage, enzymes should be removed by centrifugation. If selecting specific cells – choose selective media Embryonic tissue disaggregates more readily and yields more viable cells and proliferates more rapidly than adult tissue

Rules to follow before working with human or animal tissues Medical ethical rules or current legislation on experimentation with animals In UK, the use of embryos or fetuses beyond 50% gestation or incubation – regulated under Animal Experiments act of 1986 Work with human biopsies or fetal material – require consent from local ethical committee or relatives Favored lab materials are chick embryo and mouse embryo

What are the different techniques to get primary culture? Fine dissection to obtain explant Mechanical disaggregation involves dissection with or without maceration Enzymatic disaggregation Refer figure: 12.5 Primary explants are suitable for very small amounts of tissue. Enzymatic disaggregation gives better yield when more tissue is available and mechanical disaggregation works well with soft tissue and firmer tissue when final yield is not a problem

Primary Explant technique Developed originally by Harrison in 1907 A fragment of tissue embedded in blood plasma or lymph + embryo extract and serum Placed on a coverslip inverted over a concavity slide Clotted plasma held tissue together Outgrowth was subcultured or explant transferred Conventional microscope used to study the tissue for the appearance of outgrowth

Modified technique Tissue is chopped Washed with PBS Pieces seeded onto culture surface Medium supplemented with serum Adhere to surface and proliferate

Enzymatic disaggregation Enzymes used for disaggregation – trypsin + EDTA or only trypsin Warm trypsin - @ 37°C + removed by centrifugation + neutralized with serum and medium Cold trypsin – Soak tissue in trypsin at 4°C for 6 – 18 hrs followed by incubation at 37°C for 20-30 minutes for disaggregation CAMs, Cadherins, Integrins and transproteoglycans are disaggregated by trypsin. Harder to disaggregate adult tissues because of increase in fibrous connective tissue and extracellular matrix and reduction of undifferentiated proliferating cell pool Minimize exposure of cells to active trypsin to preserve maximum viability – follow cold trypsin procedure

Differences between warm and cold trypsin treatment Warm trypsin Shorter time period Lesser yield Centrifugation is required Cold trypsin Gives higher yield of viable cells with improved survival after 24 h culture No stirring or centrifugation is required Incubation at 4 C can be done overnight Embryonic organs

Treatment with Collagenase enzyme Used for embryonic, adult, normal and malignant Human tumor, mouse kidney, human adult and fetal brain and liver Used for too fibrous or too sensitive Requires no mechanical agitation or special equipment More than 1 gram tissue is used - collagenase can be expensive Works when trypsin cannot be used. Does not dissociate epithelial cells. Can be advantageous if you are wanting to get epithelial cells.

Mechanical disaggregation Collecting cells that spill out when tissue is sliced – Scraping/Spillage Pressing the dissected tissue through series of sieves - Sieving Forcing the tissue fragments through a Syringe Pipetting repeatedly Soft tissues – Spleen, embryonic liver, embryonic and adult brain and some human and animal soft tumors Outgrowth of cells from primary explants is a slow process and can be highly selective. Enzymatic digestion is labor intensive and proteolytic damage to cells happen. Although the digestion process gives a more closer representation to the parent cells. This method can damage cells badly because spillage and sieving are gentle methods but syringing and pipetting damage cells

Maintenance of Primary Records Keep records of culture’s origin and derivation, species, sex and tissues from which it was derived, relevant pathology, procedures used for disaggregation and primary culture Maintain records of all GLPs Hard and Soft copies Provenance of cell line (table 12.2)

This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

Disclaimer This workforce solution was funded by a grant awarded under the President’s Community-Based Job Training Grants as implemented by the U.S. Department of Labor’s Employment and Training Administration.  The solution was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor.  The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.  This solution is copyrighted by the institution that created it.  Internal use by an organization and/or personal use by an individual for non-commercial purposes is permissible.  All other uses require the prior authorization of the copyright owner.