Published: 6th Aug 2025, 17:51

Organoids. CRISPR. Immunotherapy. We bet you must have heard of at least one of these terms before. Biology is like witchcraft, but better. And being a Biologist? You don’t ask. 

Have you ever thought about how scientists turn even the smallest of the living units into something so complex and mind-boggling? It is astonishing how these little cells, if nurtured just right, can solve the greatest mysteries of life. It’s magic. 

Tissue culture is the technique of cultivating cells, tissues, or even a whole organism outside their natural environment. It is one of the most revolutionary techniques developed in modern biology. Building on the successful cultivation of microbes in laboratories, tissue culture has propelled biotechnology to new heights as it allows the cultivation of these cells and tissues in vitro (inside controlled laboratory conditions), playing a vital role in the development of vaccines and therapeutics, cloning, research, agriculture, and medicine when studying diseases like cancer. 

In this blog, we’ll dive deeper into the process of tissue culture using healthy cells, exploring how it works, why it’s important, and where it’s used. 

The Power of Starting Right: Why Healthy Cells Matter in Tissue Culture?

Before a single cell divides or a plantlet forms, there is one crucial factor at play that ensures success: checking the health of the cells the culture is started with.

What exactly do we mean when we say, “healthy cells in tissue culture”?

Just like in real life, no two cells are created equal. Scientists have to handpick the cells that show no signs of stress, mutation, or microbial infection. The choice of healthy cells to start the tissue culture is considered essential rather than just a preference and are the ones that exhibit normal growth, morphology and function. These cells don’t only grow predictably; they show consistency, behave naturally, and provide a stable, non-fluctuating foundation for experiments. 

The sources to pick out these healthy cells may vary:

• Animal cells: Cells from animal tissue are isolated to mimic human organs and are usually used in research and medicine. For example: Testing of drug susceptibility and response in animal cells.
• Plant cells: Cells from plants are generally used in tissue culture for the development of genetically stronger organisms that are more resistant to biotic or abiotic stress conditions.
• From Stem cells: Stem cells are invaluable in regenerative medicine, as they can divide and differentiate into various cell types.

Choosing healthy cells while starting a culture is also important because if the cells are compromised from the start of the experiment, everything unravels downstream. One contamination or genetic glitch and your data is all toast. 

Most of the effort while starting a healthy cell culture goes right into the earliest steps in screening, selecting and preparation. 

In the next section, we’ll step inside the lab to see how scientists guide these selected cells through the delicate, fascinating journey of tissue culture.

The Step-by-Step Process of Tissue Culture Using Healthy Cells

You are here to learn the recipe for life. From buffers to growth hormones, each ingredient serves a precise function in creating the perfect environment for healthy cells to grow, divide, and transform.  Every step hinges not just on technique, but on the quality of ingredients you put in. A detailed breakdown for the steps of tissue culture is as follows:

1. Selecting and Preparing Healthy Cells

As discussed, everything has to start with a well-chosen explant that has a viable tissue free from any signs of stress or contamination.

• Plant sources: Leaf tips, root nodules, or meristematic tissue.
  
• Animal sources: Epithelial, liver, or kidney tissues.

To maintain the cellular stability during the preparation of healthy cells, phosphate-buffered saline (PBS) or HEPES Sodium (Product code: 599) buffer may be used to rinse and hold the cells temporarily to maintain pH balance and osmotic integrity.

2. Disaggregation of Tissue

To isolate individual cells from tissue:

• Animal tissues are treated with enzymes like trypsin or collagenase, which can digest the extracellular matrix.
  
• Plant tissues are subjected to physical fragmentation and treatment with enzyme mixtures containing cellulase and pectinase help break down cell walls.

These enzymes are dissolved in isotonic solutions often buffered with HEPES Sodium (Product code: 599) or Tris to maintain cell viability throughout the process.

3. Preparing the Culture Medium

Culture medium for each type of cell has to be customized and modified according to their respective needs- this is where the science meets nutrition. 

• Animal cell media [e.g., DMEM (Dulbecco’s Modified Eagle Medium), RPMI 1640 (Roswell Park Memorial Institute Medium 1640), or MEM (Minimum Essential Medium)] often contain:
  

1. Fetal Bovine Serum (FBS) is a rich source of growth factors, hormones, and attachment proteins.
   
2. HEPES Sodium (Product code: 599) is used to stabilize pH even in open-air conditions.
   
3. Sodium bicarbonate (NaHCO₃) acts as a CO₂ buffer for pH regulation in incubators.
   
4. L-glutamine is an essential amino acid for energy metabolism and cell growth.
   
5. Antibiotics like Penicillin and Streptomycin are added to prevent microbial contamination.
   

• Plant tissue media (e.g., Murashige and Skoog (MS) medium) are supplemented with:
  

1. Macronutrients (N, P, K) and micronutrients (Fe, Zn, Mn)
   
2. Carbon source like Sucrose for energy.
   
3. Plant hormones:
   

• Auxins (like NAA or IAA) for root induction
  
• Cytokinins (like BAP or kinetin) for shoot initiation
  

4. Gelling agents like agar to solidify the medium.

4. Aseptic Handling and Sterile Setup

Sterility is the foundation of a successful culture.

1. Laminar airflow hoods provide a sterile work zone.
   
2. All media, tools, and vessels are autoclaved or filter-sterilized.
   
3. Materials like Ethanol (70%) and Sodium Hypochlorite are used to surface sterilize explants and disinfect surfaces.

5. Culturing and Incubation

Once inoculated into their respective media, the cells are incubated in specifically tailored environments:

• Animal cells are incubated at:
  
  • 37°C with 5% CO₂ to mimic human physiology.
    
  • HEPES Sodium (Product code: 599) or sodium bicarbonate maintains pH stability during CO₂ fluctuations.
    
  • High humidity prevents the evaporation of culture medium.
    
• Plant cells are incubated at:
  
  • 24–28°C in a growth chamber.
    
  • Controlled photoperiods (light/dark cycles) are maintained for photosynthesis and hormone response.

6. Monitoring and Passaging

Healthy cells grow, divide, and often need to be transferred to fresh medium:

• Inverted microscopes help track cell health and morphology.
  
• Trypsin-EDTA is commonly used to detach adherent animal cells.
  
• New flasks are re-supplied with fresh media, maintaining nutrients and buffering agents like HEPES Sodium (Product code: 599) and NaHCO₃.

7. Cryopreservation

When cells aren’t immediately needed, they’re frozen for long-term storage:

• Cryoprotectants like Dimethyl Sulfoxide (DMSO) prevent ice crystal formation that could rupture cells.
  
• Cells are stored in liquid nitrogen (-196°C).
  
• Cells are suspended in freezing media containing Fetal Bovine Serum, Dimethyl Sulfoxide, and buffered solutions to support post-thaw survival.

Why use HEPES Sodium (Product code: 599) by TM Media? 

HEPES Sodium (Product code: 599) by TM Media is widely used and trusted in cultivating cell lines by scientists. The product with CAS Number- 75277-39-3 and Chemical Formula, C8H17O4N2SNa, is used to supplement Dulbecco′s modified Eagle′s medium to culture and maintain cell lines and as a component of platelet suspension buffer. It is a white crystalline powder with a molecular weight of 260.29 g/mol. Its chemical parameters include: