Subculturing involves transferring cultured cells into fresh culture vessels to provide nutrients and space for continuous growth. It involves removing old media, washing plates, enzymatically dissociating adhered cells, and diluting the suspension into fresh media. Subculturing is necessary to maintain cell viability as cells consume nutrients over time and stagnate without fresh media. Cryopreservation preserves cells by storing them at ultra-low temperatures near liquid nitrogen, halting physical and biochemical reactions for long-term storage. Cell culture has many applications including use as disease models, cancer research, virology studies, toxicity testing, tissue engineering, vaccine production, monoclonal antibody production, and drug screening.

1. Subculturing
• The process of inoculating cultured cells into fresh culture vessels is termed as
Subculturing.
• Subculturing or splitting cells is required to periodically provide fresh nutrients and
growing space for continuously growing cell lines.
• Subculture involves removing the growth media, washing the plate, disassociating
the adhered cells, usually enzymatically and diluting the suspension into fresh
media.
• Before subculturing cells can be preserved in DMSO (di-methyl sulfoxide) or
glycerol by cryopreservation technique.

2. Why Subculturing ?
• The subculturing is necessary step to maintain the cells in continuous growth
phase.
• After some time of primary culture, the cells consume the provided nutrients and
due to lack of nutrients cells growth become stagnant and leads to death.
• Thus to maintain the cell viability, cells are transferred into fresh culture media.

3. Cryopreservation
• Cryopreservation is the process which refers to the ‘’preservation in the frozen
state’’.
• It is derived from the Greek word ‘Kryos’ meaning ‘Frost’.
• The term ‘’cryopreservation’’ means storage at very low temperature such as in
deep freezers (-80 oC) in vapor phase nitrogen (-150 oC) or in liquid nitrogen (-
196 oC).
• Cryopreservation refers to the storage of cells, tissues and organs at the ultra
low-temperature of liquid nitrogen.

4. • At such low temperatures, the stored material enters in a state of ‘’absolute
quiescence’’ as all the physical and the biochemical reactions are practically
halted.
• Thus, cryopreservation is a long term storage techniques with very low
temperatures to preserve the structurally intact living cells & tissues for
extended period of time at a relatively low cost.
• The science pertaining this activity is known as ‘’Cryobiology’’.

5. Application of Cell Culture
• Animal cell culture has several uses in pharmaceutical industry and research as
production of valuable bio molecules and in research to study cell behavior. It is
also used in the treatment of various genetic and non-genetic diseases.
 Model System:- Cell cultures provide a good model system for study-
a. Basic cell biology and biochemistry
b. The interactions between disease causing agent and cells
c. The effects of drugs on cells
d. The process and triggers for aging
e. Nutritional studies

6.  Cancer Research:– Cultured animal cells are transformed into cancer cells and
examined for therapeutic effects of any drug or chemical agent or any other
method. This studies helps in development of dose fixation studies of any
therapeutic agent.
 Virology:- Cultivation and study of viral cells is called virology. Virus are cultured
for vaccine production, genetic engineering and to study their infection
pathology.
 Toxicity Testing:- Animal cell culture is used to study the effects of new drugs,
cosmetics and chemicals on survival and growth of a number of types of cells.
Especially liver and kidney cells. Cultured animal cells are also used to determine
the maximum permissible dosage of new drugs.
 Replacement Tissue or Organ:- By using culture of stem cells, many organs can
be developed and implant in body i.e. skin, bone marrow, kidney, liver, etc.

7.  Vaccine Production:- Cells are used to produce vaccines. In industry various
microbial strains are used to grow in artificial culture media to produce vaccines,
these include polio, hepatitis, rabies, chicken pox vaccines.
 Production of Monoclonal Antibody:- Transformed B-cells are also used to
produce monoclonal antibodies for passive immunization.
 Genetic Engineering:- Cells cultured in artificial media are used for genetic
engineering or manipulation in genetic material (DNA & RNA). Genetic
engineering includes isolation, combination, transfer of genetic material into
new cells or genetically defective cells by the use of vectors and plasmids.

8.  Drug Screening and Development:- Animal cell cultures are used to study the
cytotoxicity of new drug. This is also used to find out the effective and safe
dosage of new drugs. Now these tests are being conducted in 384 and 1536 well
plates. Cell-based assay plays an important role in pharmaceutical industry.