Select Page

Cell Culture and Genetic Manipulation

by | Mar 19, 2022 | Biotechnology, Biotechnology Basics

Microbial Cell Culture and its Applications

Microbial cell culture is a technique for multiplying microbial organisms, by allowing them to proliferate in a pre-set culture medium under carefully regulated laboratory settings. Microbial cultures are fundamental diagnostic procedures widely employed in molecular biology research. The type of organism and its quantity in the sample is determined using microbial cultures. Either tested or both, It’s one of the most used microbiological diagnostic procedures, and helps find out what’s going on in people’s bodies. By allowing the infectious agent to proliferate in a specified medium, you can determine what’s causing the sickness.

In molecular biology, microbial cell cultures help in cloning and recombinant protein production. They’re also utilized in clinical settings to isolate, detect, and identify disease-causing microorganisms. Microbial culture allows cells to grow and divide in a controlled environment. To avoid contamination, bacteria and other microorganisms can be cultivated in liquid broth or solid nutritional agar culture media using aseptic procedures.

Microbial Nutrition and Culture Techniques

Understanding bacteria’s dietary needs can help with enrichment and isolation. Nutrient broths and agar plates are the most common growth media for microorganisms; nevertheless, certain germs require specific media.

Defined Media – All elements in a defined media will be in known proportions. For microbes, this entails providing the microbe with trace elements and vitamins and a set source of both carbon and nitrogen. Carbon sources include glucose or glycerol, and inorganic nitrogen sources include ammonium salts or nitrates.

Undefined Media – SSome complex components in an undefined media, such as yeast extract or casein hydrolysate, are made up of several chemical species in unknown quantities. Some bacteria have never been cultivated on specified media. Hence undefined media are sometimes chosen primarily on price and occasionally by necessity.

Enriched media provide the nutrients necessary for developing a wide range of species, including some of the pickiest. They’re frequently employed to collect as many different species of microorganisms as the specimen allows.

Fermentors / Bioreactors – These are large vessels used to cultivate microorganisms on a big scale in a controlled environment. It’s a closed vessel with enough aeration, agitation, temperature, pH control, and a drain or overflow vent for removing the cultivated bacteria and their products. The vessel can be used in an aseptic environment for several days. These also have sample ports, which allow the fermentation broth to be withdrawn at regular intervals while the fermentation is going on.

Fermentor or bioreactor designs may be divided into four categories. The most popular kind is a stirred tank bioreactor, which uses an impeller to agitate the culture media.

fermentor(Cell Culture )

Types of Microbial Culture

Batch Culture – Batch culture is the most frequent approach for studying bacterial growth in the laboratory, yet it is only one of several. A single batch of media is used to incubate the bacterial culture in a closed vessel. It starts with a small number of nutrients. After the bacterial inoculum is added to the medium, the organism will develop through the typical growth phases of lag, log (exponential), stationary, and decline. Growth is accompanied by nutrient absorption and microbial product excretion.

Continuous Culture – The growing media is constructed so that one of the nutrients is scarce. Once a result, as this nutrient is depleted, exponential growth will halt. A fresh medium containing the limiting nutrient is introduced before the nutrient is completely depleted. This cycle is repeated whenever a restricted nutrient is near to run out. An overflow device is also included in this system. This indicates that the extra volume expels the same amount of culture from the culture vessel. The culture’s production of new biomass is counterbalanced by the loss of culture from the vessel.

Fed-Batch Culture – Supplied-batch culture is a batch culture that is continually or progressively fed with fresh media without removing the growing culture. This system’s substrate concentration remains constant, but the cell density increases with time. This also implies that the volume of the culture vessel increases over time.

Measurement and Kinetics of Microbial Growth

In the presence of a proper medium and environment, microbial growth is described as an orderly rise in all chemical components. During the phase of balanced growth, the microbe’s biomass is doubled along with other quantifiable qualities, including protein, DNA, RNA, and intracellular water. In most cases, the measurement of cell mass or cell number is employed to quantify cell proliferation. The doubling time is a metric that describes microbial proliferation. During the organism’s balanced growth (i.e., log phase), it is the period necessary for the cell mass or number to double its initial value.

One of the simplest techniques to monitor microbial growth is to count the number of cells.

(1) It is done by extracting the cells from the medium and drying them until a consistent weight is attained, then measuring the dry weight of the cell material in a set volume of the culture. The dry cell weight of 1 billion E. coli cells, a commonly used bacteria in labs, is roughly 150 mg.

(2) The absorbance of cell suspensions in a spectrophotometer also quantifies cell proliferation. The fact that tiny molecules scatter light according to their concentration supports this concept.

We’ll use bacterial binary fission as an example to better understand microbial growth dynamics, where each cell division creates two identical daughter cells. The time it takes a cell to divide is called generation time. Because the population of cells doubles throughout this period, the generation time is also known as doubling time (td).

One E. coli cell in nutritive media, for example, will divide every 20 minutes. One cell will have grown to eight cells after one hour of development (three generations) (1 to 2, 2 to 4, 4 to 8). The growth in cell number over time is exponential or logarithmic because the number of cells doubles with each division.

The microbial population is nearly stable during the lag period. However, when the bacteria adjust to the culture conditions, this is a phase of high metabolic activity. Cell division happens with increasing frequency as the cells have acclimated to the culture until maximal growth is attained. This is referred to as the log phase. Exponential growth occurs at this phase, with cell biomass or cell population increasing constantly.

inoculation(Cell Culture )

Isolation of Microbial Products

After the fermentation is finished, the required metabolite must be recovered. Separation of the cells from the fermentation broth will be the bare minimum. Purifying the metabolite with or without cell disruption is also possible; cell disruption is required if the metabolite is intracellular. Downstream processing is the term for these procedures. Isolation of the desired microbial product involves the following steps:

  1. separation of cells from the fermented broth
  2. cell disruption if the product is intracellular, or broth concentration if the product is extracellular.
  3. initial metabolite purification
  4. metabolite-specific purification in which the metabolite of interest is purified to a high degree

Stage 1– The separation of entire cells (cell biomass) and other insoluble substances from the culture broth is the first stage in product recovery. Separating solids from liquids can be done in a variety of ways. Flotation, flocculation, filtering, and centrifugation are examples of these processes.

Stage 2– Several biotechnological products are found within the cells, as previously indicated. Such molecules must first be released to be further processed and isolated. Physical, chemical, or enzymatic processes can disintegrate or disturb bacteria or other cells. Because the property of cell disruption or breaking varies greatly, the choice of a technique is dependent on the type of the cells.

Stage 3- Water makes up 80-98 percent of the filtrate devoid of suspended particles (cells, cell debris, etc.). The intended product is a non-essential component. To achieve the product concentration, the water must be removed. Evaporation, liquid-liquid extraction, membrane filtration, precipitation, and adsorption are all common methods for concentrating biological products. The process used is determined by the type of the intended product and the cost factor.

Stage 4– Chromatography effectively purifies the biological products of fermentation (proteins, pharmaceuticals, diagnostic compounds, and research materials). It’s essentially an analytical technique for isolating closely related compounds from a mixture. A stationary phase and a mobile phase are often used in chromatography.

Stage 5– The term “formulation” refers to the process of keeping a biotechnological product active and stable during storage and delivery. Low molecular weight goods can be manufactured by concentrating them and removing most of the water. Formulation of certain tiny molecules (antibiotics, citric acid) can be accomplished via crystallization with the addition of salts.

Strain Isolation and Improvement

The microbe-containing material (for example, soil) is placed in a nutritive medium and grown in shaking cultures. The growth parameters (e.g., temperature) and nutrients in the medium are set up so that bacteria of interest may thrive. This is referred to as the enrichment technique.

Enrichment culture is a technique for isolating an organism of interest from the rest of the world so that it can flourish in ideal circumstances while being isolated from any competition.

The enhanced culture can be sub-cultured by taking a little inoculum and placing it in a new medium. As a result, the intended organisms’ development improves with time. Further testing is carried out utilizing how the organism demonstrates the desired qualities. For example, if we’re seeking a microbe that makes antibiotics, we may test for it by cultivating the culture on an agar plate in the presence of the bacteria we’re looking for antimicrobial action against. Immunological approaches are also available, in which particular antibodies are used to detect the bacteria that produce the compounds.

The strain isolation process only finds a strain with the capacity or aptitude to synthesize a desired chemical. It does not guarantee that it will create enough molecules to be commercially feasible. Traditional genetics and genetic engineering techniques are applied to increase the strain’s desirable features.

After obtaining a strain that produces a unique or desirable product, it must be properly conserved for future use. If not done correctly, the strain may become extinct or exhibit a reduction in the production of the product for which it was separated.

Agar: Cultures are cultivated on agar slants or stabs and kept at temperatures ranging from 5 to -20°C.

Liquid Nitrogen: The culture is cultivated, and a cryoprotective substance such as glycerol (10-30%) is added before being stored in liquid nitrogen. These are frozen in liquid nitrogen and delivered in sealed ampoules. (-176 to -196 degrees Celsius)

Lyophilization: often known as freeze-drying, is the process of freezing a culture and then drying it under a vacuum. Sublimation of cell water occurs as a result of this.

Applications of Microbial Culture Technology

Microbial cultures offer a lot of promise when it comes to producing valuable substances. Once the microbial culture has been created, it may be utilized to produce a variety of chemicals based on its metabolic activity.

The most ancient application of microbial cultures is in the manufacture of fermented foods like curd and cheese, in which entire bacteria are utilized as starting cultures. At the same time, microorganisms are also employed in the production of bacterial vaccines, such as typhoid and TB vaccines. Another example of using entire microbes as a source of protein is a single-cell protein (SCP).

Primary metabolic products such as alcohol and acids are examples of primary metabolic products, whereas secondary metabolic products such as antibiotics are examples of secondary metabolites generated by various microbes. Microbial metabolism has also been used in the manufacture of vitamins by microbes. Microorganisms have become more essential as hosts for creating recombinant proteins utilizing genetic engineering techniques in recent years. Human insulin expression in E. coli and hepatitis B surface antigen expression in yeast for hepatitis B vaccine production are two significant instances of microbe applications for human usage.

Microbial metabolism is also employed to transform inappropriate substrates to usable products in extracting metals from ores and the treatment of liquid waste. To generate metabolites, microbial cultures can be used in six distinct methods in general. They are as follows:

  1. Complete microbial cell production (for food, vaccines).
  2. Synthesis of primary metabolites (acids, alcohol).
  3. Synthesis of secondary metabolites (antibiotics).
  4. Reactions of biotransformation (enzymatic, steroid).
  5. The use of metabolism as a tool (microbial leaching, biodegradable waste treatment).
  6. Protein synthesis using recombinant DNA (therapeutic proteins).

Plant Cell Culture

Plant cell, tissue, and organ culture is a collection of procedures to grow and multiply cells and tissues in an aseptic and controlled environment utilizing nutritional solutions. This technique investigates in vitro settings that enhance cell proliferation and genetic reprogramming. Plant tissue culture, which was first created in the early 1960s, has now become a routine method in modern biotechnology.

Plant tissue culture is a set of procedures for maintaining or growing plant cells, tissues, or organs in sterile circumstances on a known-composition nutrient culture medium. Micropropagation is a way of producing clones of a plant that is commonly utilized. Different plant tissue culture techniques may have benefits over traditional propagation methods.

Cell and Tissue Culture Techniques

Tissue culture is a method of biological study in which animal or plant tissue pieces are moved to an artificial environment where they can survive and function. A single cell, a population of cells, or a full or part of an organ can all be cultured.

Virtually any plant portion (known as an explant) or cells can be used to regenerate the entire plant. The following are the steps in the basic plant tissue culture technique:

  • Explants such as shoot tips, leaves, cotyledons, and hypocotyls were chosen.
  • Surface sterilization of the explants using disinfectants (e.g. sodium hypochlorite), followed by sterile distilled water washing.
  • Explants are inoculated (transferred) into an appropriate nutritional medium in culture containers under sterile conditions (i.e., in a laminar flow cabinet).
  • Growing the cultures in a growth chamber or plant tissue culture room with the proper physical conditions.
  • Elongation and regeneration of shoots from cultured plant tissues.
  • Regenerated shoots are rooted in rooting media.
  • Following the acclimatization (tissue hardening) of the regenerated plants, the plants are transferred to the transgenic green-house or field environments.

Plant tissue preparation for tissue culture is done in an aseptic environment with HEPA filtered air delivered by a laminar flow cabinet. The tissue is then cultivated in sterile containers such as Petri plates or flasks in a regulated temperature and light environment. Because living plant materials from the environment are naturally contaminated with microorganisms on their surfaces (and sometimes interiors), their surfaces must be sterilized in chemical solutions before suitable samples (known as explants) can be taken.

When cell suspension cultures are required, the sterile explants are normally put on the surface of a sterile solid culture media, but they can also be placed straight into a sterile liquid medium. Inorganic salts and a few organic minerals, vitamins, and plant hormones make up most solid and liquid media. Solid media combine liquid media with a gelling ingredient, commonly pure agar.

Applications of Cell and Tissue Culture

Plant tissue culture is widely utilized in horticulture, forestry, and plant sciences. The following are some examples of applications:

  • The use of meristem and shoot culture creates vast numbers of identical individuals in the commercial production of plants used as potting, landscaping, and florist subjects.
  • Plant species that are uncommon or endangered are being protected.
  • Tissue culture may be used by a plant breeder to screen cells rather than plants for desirable characteristics, such as herbicide resistance/tolerance.
  • Plant cells are grown in liquid culture in bioreactors on a large scale to produce useful substances such as plant-derived secondary metabolites and recombinant proteins that are utilized as biopharmaceuticals.
  • Protoplast fusion and regeneration of a unique hybrid between distantly related species.
  • In vitro selection for stress-resistant plants, for example, to quickly examine the molecular basis for physiological, metabolic, and reproductive systems in plants.
  • To cross-pollinate closely related species and then tissue culture the resultant embryo, which would otherwise perish (Embryo Rescue).
  • For chromosomal doubling and polyploidy induction, such as doubled haploids, tetraploids, and other polyploids. Antimitotic drugs such as colchicine or oryzalin are commonly used to achieve this.
  • As a transformation tissue, followed by either short-term testing of genetic constructs or transgenic plant regeneration.
  • Sugarcane, potatoes, and a variety of soft fruit species can all benefit from approaches like meristem tip culture to develop clean plant material from viruses stock.
  • It is possible to create identical sterile hybrid species.
  • Artificial seed synthesis on a large scale via somatic embryogenesis

Transgenic Plants with Beneficial Traits

Over the last several decades, significant progress has been achieved in understanding gene function, isolating novel genes and promoters, and using these genes to generate transgenic or genetically modified (GM) crops with enhanced and innovative characteristics.

Crop plants may be extremely prolific when grown under optimal conditions, but ideal growing conditions are unusual. Furthermore, plants are subjected to biotic (viral, bacterial, fungal diseases, nematodes, insect pests, and weeds) and abiotic (salinity, drought, severe temperatures, nutrient shortage, and so on) stressors, which result in massive losses in crop production and quality.

Chemical and biological insecticides and the usage of resistant cultivars have only had limited success and have their own set of constraints. To accommodate the demand, newer and more effective technologies are required. Biotechnological techniques can be utilized in this context to develop transgenic plants that are more resistant to diseases, pests, and abiotic stressors.

Disease Resistance: Pathogens (viruses, fungi, and bacteria) attack agricultural plants, reducing production and quality significantly. Farmers have traditionally used chemical pesticides or resistant crop types produced by breeders, but both strategies have drawbacks. The production of disease-resistant transgenic crop plants by transferring resistance genes from various sources is an alternate and valuable technique.

Pest Resistance: Many insects and nematodes attack all agricultural plants, reducing production and quality considerably. Farmers employ synthetic pesticides extensively to reduce these losses (both food and money), which have serious health and environmental consequences. Transgenic technology offers a new and unique way to enhance pest control management that is environmentally benign, effective, long-lasting, and yield-enhancing. Cry genes (also known as Bt genes) from the bacterium Bacillus thuringiensis were the first genes accessible for genetic engineering of agricultural plants for pest resistance.

Fruit Ripening: Ethylene, a gas hormone, is involved in fruit ripening control. As a result, ripening can be halted by preventing or lowering ethylene production. This can be accomplished by inserting ethylene-forming gene(s) into the crop plant in a way that suppresses their expression. Fruits from such trees ripen slowly (although maybe ripened with ethylene) and are ideal for long-distance transport without rotting, as they have a longer shelf life due to their gradual ripening.

Vitamin A: Vitamin A is obtained either directly from animal foods or indirectly from carotenoids found in green leafy vegetables and fruits, converted to vitamin A in the body. Golden Rice seeds are golden in color because pro-vitamin A is created throughout the grain. Golden Rice was genetically modified by inserting three extra genes for iron supply and absorption from diverse species. Transgenic agricultural plants are also being developed to increase the levels of other vitamins, including vitamin E (an important antioxidant in humans) and vitamin K.

Animal Cell Culture

Animal cell culture is a biotechnological approach that involves artificially growing animal cells in a suitable environment. These cells are typically derived from multicellular eukaryotes and their established cell lines. Animal cell culture is a commonly used technique for isolating cells and cultivating them in artificial environments.

This approach was originally designed as a laboratory procedure for specific investigations, but it has now been modified to keep living cell lines isolated from their source. The invention of the basic tissue culture medium, which allows a wide range of cells to operate under diverse circumstances, has aided the development of animal cell culture techniques.

Identifying various roles and processes of operation of distinct cells has been aided by in vitro cultivation of isolated cells from various animals.

Cancer research, vaccine manufacture, and gene therapy are some of the areas where animal cell culture has found the most use. Growing animal cells on the artificial medium are more challenging than growing microorganisms on artificial media. Thus more nutrients and growth agents are required.

Animal cell cultures may be made from various cell types, and complex structures such as organs can be utilized to start organ culture in vitro. Depending on the technique’s aim and application, cells, tissues, or organs can all be employed in the culture process.

Animal Cell Culture Techniques

Depending on the number of cell divisions that occur during the procedure, animal cell cultures may be categorized into two types.

Primary Cell Culture: Primary cell culture is the initial culture generated directly from animal tissue by mechanical and chemical disintegration or enzymatic procedures. The slow-growing cells in primary cell culture have all of the features of the original tissue or cells. These cultures have the same number of chromosomes as the original cells since they were taken straight from the source. Primary cell cultures are used to retain and sustain cell development in an artificial growth medium under certain conditions.

Secondary Cell Culture: After the primary cell cultures have been subcultured in a new culture medium for a long time, secondary cell cultures are obtained. Secondary cell cultures have longer-lasting cells due to the availability of sufficient nutrients at regular intervals. Primary cell cultures are preferred over secondary cell cultures because secondary cell cultures are more commonly accessible and easier to produce and store. These are made by enzymatically treating adherent cells, then washing and resuspending the cells in specific quantities of the new medium.

Cell Lines

A cell line is a collection of cells derived from a subculture of pure cell culture in primary culture. Cell lines normally have functional characteristics that are similar to primary cells, although their genotype and phenotype can be altered.

Based on the development patterns of the cells, cell lines may be further split into two groups:

  • Finite Cell lines – Finite cell lines are cell lines in which the cells in the culture divide a limited number of times before dying. The cells of the limited cell lines can divide 20 to 100 times before dying and no longer being able to divide.
  • Continuous Cell lines – Continuous cell lines are cells that continue to proliferate indefinitely after being a subculture. The cells in continuous cell lines multiply more quickly, forming an independent culture. They may divide endlessly and are immortal. The cells in the continuous cell lines are tumorigenic and can be converted by genetic changes.

Growth Conditions

  • Animal cell culture necessitates using a more complicated and specialized culture medium than the basic culture media used for microbial development.
  • Inorganic salts, nitrogen supply, energy source, vitamins, fat and fat-soluble vitamins, growth factors, and hormones are some of the most significant fundamental components of the media. pH buffering systems and antibiotics may be used in some circumstances.
  • Because various species require different temperatures for cell growth and division, the temperature for growth is determined by the cell source.
  • Warm-blooded animal cells should be cultivated at 37°C, whereas cold-blooded animals should be grown between 15°C-25°C.

Applications of Animal Cell Culture

Animal cell culture and recombinant DNA technologies have manufactured several therapeutically relevant protein pharmaceuticals. The following are a few examples of animal cell culture applications:

Vaccine Manufacturing: The use of animal cell culture in creating viral vaccines is a common practice. A recombinant vaccine against hepatitis B and poliovirus was developed using this approach. Immortalized cell lines are utilized for large-scale or industrial manufacturing of viral vaccines.

Gene Therapy: The advancement of animal cell culture is crucial for gene therapy advancements. To eliminate flaws and illnesses, cells with defective genes can be replaced with a functioning gene.

Biopesticide Production: Because of their quicker development rate and higher cell density, animal cell lines like Sf21 and Sf9 can be utilized to make biopesticides. Animal cell culture may also be used to generate organisms like baculovirus.

Recombinant Protein: Recombinant therapeutic proteins such as cytokines, hematopoietic growth factors, growth factors, hormones, blood products, and enzymes may all be made in animal cell cultures. Baby hamster kidney and CHO cells are two of the most prevalent animal cell lines utilized to make these proteins.

Cancer Research: Cancer cells can also be grown; therefore, animal cell culture may be utilized to explore the distinctions between cancer cells and normal cells. The distinctions enable more in-depth research into the causes and consequences of various carcinogenic chemicals. Normal cells may be cultured using chemicals, viruses, and radiation to become cancer cells.

Model System: Cells generated by cell culture can be used as a model system for research into cell biology, host-pathogen interactions, medication effects, and the impact of changes in cell composition.

Stem Cell Technology

Stem cell technology is a fast-evolving science that brings together the efforts of cell biologists, geneticists, and physicians to give promise for the successful treatment of a range of cancers and non-cancerous disorders.

These are totipotent progenitor cells that can self-renew and differentiate into many lineages. They are good candidates for in vitro modification because they survive well and divide consistently in culture.

Stem cells are progenitor cells that can self-renew and differentiate into various cell types.

Many malignant and non-malignant disorders might benefit from stem cell therapy. Peripheral blood stem cells are commonly employed in both autologous and allogeneic bone marrow transplantation.

Treatment of hereditary or acquired disorders may be possible by gene transfer into hematopoietic stem cells. Embryonic stem cells may be cultivated in vitro to form sophisticated organs in the future.

Neuronal stem cells are being employed to replace neurons in neurodegenerative illnesses, including Parkinson’s and Huntington’s.

The generation of blood cells has been the subject of the greatest research (hematopoiesis). It was previously recognized that hematopoiesis occurs in mice’s spleen and bone marrow. Each kilogram of body weight per day, 100,000 hematopoietic stem cells create one billion RBC, one billion platelets, one million T cells, and one million B cells. Ernest McCulloch and James Till founded the area of stem cell research at the University of Toronto in the 1960s.

ES cells, which are separated from the inner cell mass of blastocysts, and adult stem cells, which are present in adult tissues, are the two forms of mammalian stem cells. Pluripotent stem cells (ES cells) can develop into various specialized tissues. Adult stem cells are multipotent (lineage limited) and serve as a bodily repair mechanism by ensuring the appropriate turnover of regenerative organs, including blood, skin, and intestinal tissues. Through cell culture, stem cells are now regularly produced and converted into specialized cells such as muscles or nerves and employed in medicinal therapy.

 

 

Author

  • The author has done a master's in Environmental science and is currently working as chief Environmental Advisor with New Delhi State Government.

0 Comments

Submit a Comment

Your email address will not be published.