1. Primary Culture
Chapter 12

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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

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

11. 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 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

12. 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

13. 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.

14. 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

15. 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)

16. 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 ). 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.

17. 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.