Tissue Culture Technique

 1. Introduction

Tissue culture is the process of growing cells, tissues, or organs in an artificial environment under controlled laboratory conditions. It plays a fundamental role in modern biological sciences, biotechnology, and medicine, allowing researchers to study cellular processes, gene expression, and the effects of drugs or toxins on living systems. The basic principle involves maintaining cells in sterile conditions with an appropriate supply of nutrients, gases, temperature, and humidity that mimic the body’s natural physiological environment.

2. Historical Background

The development of tissue culture began in 1907, when Ross Granville Harrison successfully cultured frog nerve fibers in a lymph medium the first demonstration that cells could survive and grow outside the organism. Alexis Carrel (1912) later cultured chick heart tissue, demonstrating the potential of maintaining tissues for long durations. A revolutionary advancement occurred in 1951, when HeLa cells were derived from Henrietta Lacks, a cervical cancer patient. These were the first immortal human cell line capable of indefinite proliferation in vitro. Their robustness made them a cornerstone for discoveries in virology, cancer biology, and genetics.

3. Types and Classification of Cells in Tissue Culture

Cells used in tissue culture are commonly classified based on two criteria: (1) Origin or Lifespan (Primary vs. Secondary Cells) and (2) Growth Behavior (Adherent vs. Suspension Cells). These categories often overlap and influence each other, determining the culture’s requirements and maintenance strategies.

3.1 Classification Based on Origin or Lifespan

a. Primary Cells

Primary cells are freshly isolated from living tissues through enzymatic digestion (e.g., trypsin, collagenase) or mechanical disaggregation. They closely mimic the morphology and physiology of the original tissue and provide the most biologically relevant model. However, they have a limited lifespan and undergo a finite number of cell divisions before senescence. Examples include hepatocytes, keratinocytes, and neurons.

b. Secondary (Continuous) Cells

Secondary or continuous cells originate from primary cultures that have acquired the ability to proliferate indefinitely. This may occur spontaneously or through transformation by viruses or chemical agents. These immortalized cell lines are easy to maintain, reproducible, and cost-effective for research and industrial purposes. Examples include HeLa, HEK293, and CHO cells.

3.2 Classification Based on Growth Behavior

a. Adherent Cells

Adherent cells need a surface for attachment, where they spread and form a monolayer. They are detached for subculturing using trypsin-EDTA and grown in T-flasks or culture plates. Examples include fibroblasts, epithelial cells, and endothelial cells.

b. Suspension (Floating) Cells

Suspension cells grow freely in liquid medium and do not require surface attachment. They are ideal for large-scale cultures and bioreactors. Examples include lymphocytes, hybridomas, and leukemia cell lines.

Table 1: Types of Cells

Category	Growth Behavior	Examples	Key Features
Primary Adherent Cells	Attached to surface	Fibroblasts, hepatocytes	Closely resemble native tissue; limited divisions
Primary Suspension Cells	Floating in medium	Lymphocytes, blood cells	Freshly isolated, short lifespan
Secondary Adherent Cells	Attached to surface	HeLa, HEK293	Immortalized, robust growth
Secondary Suspension Cells	Floating freely	Hybridoma, K562	Large-scale culture, indefinite growth

 Consumables and Equipment Used in Tissue Culture

Tissue culture requires high-quality, sterile, and biocompatible materials to maintain cell viability and avoid contamination. Common consumables include T-flasks, pipettes, culture plates, Falcon tubes, cryovials, and filters. Essential equipment includes CO₂ incubators, laminar flow hoods, centrifuges, water baths, and microscopes.

 Table 2A: Consumables used in the Tissue Culture 

Consumable	Material	Purpose/Use	Notes
T-Flasks (T25, T75, T175)	Polystyrene (treated)	Cell growth for adherent cells	Treated surface enhances attachment
Pipettes (1–25 mL)	Polystyrene	Sterile transfer of liquids	Single-use, sterile
Multiwell Plates (6–96 wells)	Polystyrene	Culturing, screening, assays	May be coated for adhesion
Falcon Tubes (15/50 mL)	Polypropylene	Centrifugation, sample mixing	Temperature and chemical resistant
Cryovials	Polypropylene	Storage of frozen cells	Screw cap with O-ring seal
Filters (0.22/0.45 µm)	PES or cellulose acetate	Media sterilization	Prevents microbial contamination
Serological Pipettor	—	Liquid transfer control	Used with sterile pipettes
Pipette Tips	Polypropylene	Precise volume transfer	Sterile and disposable
Cryogloves	Insulated polymer	Handling liquid nitrogen materials	Protects hands from cold burns
Cryoboxes / Racks	Polycarbonate	Organizing cryovials	Fit into nitrogen tank racks

 

Table 2B: Equipment Used in Tissue Culture

Equipment	Function/Purpose	Description/Notes
Laminar Flow Hood / Biosafety Cabinet (Class II)	Provides sterile workspace	HEPA-filtered airflow prevents contamination
CO₂ Incubator	Maintains temperature (37°C), humidity, and CO₂ levels (5%)	Essential for mammalian cell growth
Inverted Microscope	Visualizes cells in culture flasks or plates	Lens positioned below stage
Water Bath (37°C)	Thawing frozen cells and warming media	Must be regularly cleaned
Centrifuge	Pellet cells, remove supernatant	Speed and time vary by cell type
Refrigerator (4°C)	Stores reagents and media	Prevents spoilage of chemicals
Freezer (-20°C)	Stores enzymes, antibiotics, and sera	Medium-term storage
Deep Freezer (-80°C)	For temporary cell preservation	Pre-freezing before liquid nitrogen
Mr. Frosty Freezing Container	Controls freezing rate (1°C/min)	Used before transfer to nitrogen tank
Liquid Nitrogen Tanks	Long-term storage of cryopreserved cells	Types: Dewar, vapor-phase, and liquid-phase
Cryogenic Storage Rack	Organizes cryovials inside nitrogen tank	Prevents vial loss
pH Meter	Monitors medium pH	Ensures physiological balance
Autoclave	Sterilizes glassware and tools	Uses steam under pressure

 Reagents Used in Tissue Culture

Essential reagents provide nutrients, buffering, and protection to cells during culture and preservation. Examples include DMEM, FBS, HEPES buffer, Trypsin, and Penicillin-Streptomycin.

Table 3: Reagents Used in Tissue Culture

Reagent	Full Name / Description	Purpose
DMEM	Dulbecco’s Modified Eagle Medium	Nutrient-rich basal medium
RPMI-1640	Roswell Park Memorial Institute Medium	Used for suspension cells
FBS	Fetal Bovine Serum	Provides growth factors and hormones
MEM	Minimum Essential Medium	Basic nutrient solution for adherent cells
HEPES	Buffering reagent	Maintains pH stability
Penicillin-Streptomycin	Antibiotic mix	Prevents bacterial contamination
Trypsin-EDTA	Enzyme solution	Detaches adherent cells
PBS	Phosphate Buffered Saline	Rinsing and washing cells
DMSO	Dimethyl Sulfoxide	Cryoprotectant during freezing
Bulbecco	Alternative basal medium	Cell growth support

 Cell Counting

Accurate cell counting is crucial to assessing cell density and viability before subculturing. Trypan Blue is used to differentiate live and dead cells using a hemocytometer under a microscope.

Table 4: Items used in the counting of cells in tissue culture

Tool/Reagent	Purpose	Description
Hemocytometer (Neubauer Chamber)	Manual cell counting	Uses grid lines for counting cells
Microscope	Visualization	Used to view cells and viability
Trypan Blue	Stains dead cells blue	Live cells exclude dye
Automated Cell Counter	Quick digital counting	Uses imaging or electrical impedance
Eppendorf Tubes	Mixing samples	Used for Trypan Blue mixing
Pipettes & Tips	Accurate sample handling	For transferring cell suspensions

Thawing of Cells

Thawing must be performed carefully to minimize thermal shock and ensure maximum viability. Frozen vials are placed in a 37°C water bath, then transferred to culture media to remove DMSO.

Process:

1. Remove the cryovial from liquid nitrogen.
2. Quickly thaw in a 37°C water bath until a small ice crystal remains.
3. Transfer to pre-warmed culture medium.
4. Centrifuge to remove DMSO.
5. Resuspend cells and incubate at 37°C with 5% CO₂.

Table 5: Thawing Materials and Purpose

Material/Equipment	Purpose	Precaution
Water Bath (37°C)	Rapidly thaw cells	Avoid overheating
DMSO	Cryoprotectant	Must be removed post-thaw
Centrifuge	Removes DMSO and debris	Use gentle spin
CO₂ Incubator	Cell recovery after thaw	37°C, 5% CO₂, high humidity
Pre-warmed Media	Revives cells	Maintain sterility
Cryogloves	Safe handling of cryovials	Prevent frostbite

 Cryopreservation

Cryopreservation allows long-term storage of cells at -196°C in liquid nitrogen using DMSO as a cryoprotectant. Mr. Frosty provides controlled freezing at 1°C per minute before liquid nitrogen storage.

 Table 6: Reagents and items used in the cryopreservation

Component	Purpose	Temperature / Notes
DMSO	Prevents ice crystal formation	Used at 10% concentration
FBS	Nutrient support during freezing	Enhances survival rate
Cryovials	Stores frozen cells	Screw cap, leak-proof
Mr. Frosty	Controls freezing rate	Used before LN₂ transfer
Liquid Nitrogen Tank	Long-term storage	−196°C
Cryogloves / Cryogoggles	Safety during handling	Protects from frostbite
Freezing Box / Rack	Organizes vials	Fits tank canisters

 Conclusion

Tissue culture technology has transformed biological and medical research. Understanding classifications, consumables, reagents, and preservation ensures reproducibility and success in laboratory studies.