Animal Cell Culture Techniques : Advances and Recent Trends
Animal Cell Culture
Cell culture refers to the removal of cells from an animal, plant or insect followed by their subsequent growth in a favorable artificial environment.
Animal Cell Culture, as the name suggests, involves extraction of cells from the tissues or organs of animals to study cell growth and differentiation, identification of growth factors, and understanding of mechanisms underlying the normal functions of various cell types.
The source of these cells could be either:
− Direct removal from the tissue followed by dis-aggregation using enzymatic or mechanical means before cultivation,
− Or, they could be derived from already established cell lines and cell trains.
Examples of commonly used animal cells include: lymphocytes, cardiac, epithelial or skeletal cells.
In the past century, animal cell culturing has had major applications in the production of therapeutic recombinant proteins & hormones, monoclonal antibodies, recombinant viral vaccines (against Hepatitis B, HPV), gene therapy, biopesticides and the recent construction of organ/ tissue culturing chips. Hence, it becomes extremely important to understand the core processes underlying such major developments.
A Brief History of Milestones in Animal Cell Culture
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| A Brief History of Milestones. Illustration ©2021 Imber Science |
Recent Milestones (In the Last Two Decades)
1998 – Thomson & Gearhart isolate and culture human embryonic stem cells
2002 – Atala & Lanza exploit tissue engineering
2002 – Cloneaid claimed to produce cloned human baby named EVE.
2003 – Zheng created Protein crystallization chip
2004 – FDA approves first anti-angiogenic monoclonal antibody that inhibits the growth of blood vessels or angiogenesis (for cancer therapy).
2006 – Yamanka obtains induced Pluripotent Stem cells (iPS)
2010 – Atala demonstrates 3D tissue and organ bioprinting techniques
2012 – Maria Blasco performed first gene therapy successful against aging-associated decline in mice.
Current Scenario In Animal Cell Culture
Types of Animal Cell Cultures
Depending on the cells that have been used to culture, the cell culture could be classified into two types:
1. Primary Cell Culture
Refers to the condition in which cells are either extracted directly from the organ/tissue of the organism (mechanical method) or obtained indirectly by the enzymatic or chemical means and proliferated under appropriate conditions on suitable containers (in vitro) until they occupy all the available substrate, i.e. reach a state of confluency. At this stage, it becomes indispensable to transfer the cells to a new container with fresh growth medium to enable their continuous growth. (Subculturing)
Primary cells are not only morphologically similar to parent cells, but also are capable of only limited number of cell divisions after which they enter a non-proliferative state called senescence and eventually die out.
On the basis of their growth, the primary cells are further classified as:
a) Adherent or anchorage dependent primary cells: Cells that require a solid surface for growth and attachment and propagate as a monolayer, attached to the container. The attachment is essential for proliferation and often the cells are susceptible to contact inhibition. That is, the growth ceases once they have overgrown the culture vessel. Majority of tissue derived cells are anchorage-dependent. Eg: Human Fibroblasts and epithelial cells.
b) Anchorage independent or Suspension cells:Do not require a substratum for attachment and can grow in liquid nutrient medium. Eg: Hematopoietic cells (from bone marrow, spleen, or blood) and cells/cell lines derived from malignant tumors.
Primary cell cultures are often preferred over continuous cell lines in experimental systems where they are considered to be more physiologically similar to in vivo cells. However, culturing primary cells is more difficult than the culturing of established cell lines.
2. Secondary Cell Culture
Obtained from sub-culturing of Primary Cells, which often causes phenotypic and genotypic uniformity of the cell population (Homogenous population). It further prevents senescence caused due to prolonged high cell density.
Depending on the life span of the cell, cell lines are categorized into:
a. Finite cell lines: formed by the first sub-culturing of Primary cells and possess limited life span with limited number of cell divisions after which they senesce and die out. However, the proliferative potential of some human finite cell lines could be extended by introducing viral transforming genes (like SV40 transforming-antigen genes) They express a phenotype intermediate to the finite and continuous cell lines. Used for generating stably transfected clones.
b. Continuous cell lines: Possess an indefinite passaging value and number of cell division capacity. These are produced due to a stable, heritable mutation in the finite cell lines which gives rise to unlimited proliferative potential (called in vitro transformation / immortalization) Eg: Cultures derived from cancerous cells.
Types of Media & Suspensions Used
Media
In order to maintain a healthy, continuous growth of cells, it’s essential that the conditions simulated in vitro (of temperature, nutrients, pH, osmolarity, viscocity) are almost identical to the ones in vivo(compounded with a sterile environment). Hence, it becomes important to select a nutrient medium that supports the optimum proliferation of the cells and complies with the need of the experiment. Culture media could be divided into:
1. Natural Media
Comprise the naturally occurring fluids:
a. Biological Fluids: plasma, serum, lymph, human placental cord serum, amniotic fluid, pleural fluid, fetal calf serum, insect haemolymph.
b. Tissue Extracts: Extract of liver, tumours, leucocytes, spleen and bone marrow, extracts of bovine and chick embryo.
c. Clots: Plasma clots and coagulants
2. Artificial Media (Synthetic media)
Produced by adding supplemental nutrients to meet the specific requirements of the cells used, so that an ideal environment is created.
On the basis of specificity of need of cells, media can be grouped into:
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| Animal Cell Culture Techniques: Types of Media Illustration. ©2021 Imber Science |
Cell Suspensions
− Refer to a type of culture where cells are suspended in liquid medium.
− Obtained by breaking up of a callus in an agitated liquid medium (agitation enables gaseous exchange), thus releasing single cells which are transferred to a fresh medium.
− Cell Suspensions are advantageous in the sense that they allow the cells to be uniformly bathed, aerated and easily manipulated.
Morphology of Cells
Cells in culture could be divided into three types on the basis of their morphology. Given below:
(Pictures’ Source: ThermoFischer SCIENTIFIC)
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| Types of Cells on the basis of Morphology. Illustration ©2021 Imber Science |
Trypsinization
In Sub-culturing, each time a few cells need to be transferred into a new vessel with fresh medium, they need to be disaggregated from the neighbouring cells as well as the surface medium. Disaggregation becomes even more important in Adherent cell cultures, where proteins secreted by the cells form a tight bridge between the cell and the surface. Proteins are broken down at specific positions using a mixture of trypsin-EDTA. Trypsin is believed to be either protein-degrading or proteolytic and brings about further hydrolysis of pepsin-digested peptides (process called Trypsinization.)
Trypsinization could be either warm (at 37°C) or cold (pre-exposure of tissue to ice). Cold Trypsinization is used in cases where the cells are more prone to damage by prolonged exposure to warm trypsin. (37°C)
The Process of Trypsinization for Adherent Cells is given below. (Source: Verma et al., 2014)
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| Animal Cell Culture Techniques: Process of Trypsinization |
Modern Techniques in Cell Culture
3D Cell Culturing
Traditional Cell Culturing which involves growth of a monolayer of cells on a flat and rigid surface are two-dimensional (2D), a major disadvantage of which is loss of tissue specific architecture, mechanical & biochemical signals and cell-to‐cell interactions. Under in vivo conditions, cells consume oxygen and other nutrients in a much more dynamic way and these conditions fail to be mimicked by conventional 2D cell culturing. This is where 3D cell culturing comes into picture, which provides a better, physiologically active and closer biomimetic environment of cells in vivo which are continuously bathed by the bloodstream and preserves the interactions between cell-to-cell, cell-to-matrix and extracellular matrix (ECM) components.
3D Cell culture models could be grouped in two major categories:
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| Animal Cell Culture Techniques: Types of 3D Cell Culture Models. Illustration ©2021 Imber Science |
1. Scaffold Based
Though this technique has gained momentum in the past few years, Carrel for the first time, used silk threads as scaffolds to avoid necrosis of his cell colonies in the central region, about a hundred years ago. Modern technologies are more or less based on this same method which stimulate cells to grow in a dynamic 3D environmentusing an external physical support.
a. Polymeric Hard Scaffolds
Pre-fabricated scaffolds or matrices arecreated using polymers like Polystyrene (PS) and Polycaprolactone (PCL). These could be created as a porous disc (A), an electrospun (B), or an orthogonal layering scaffold (C). The cells can attach, migrate, and fill in the gaps within the scaffold to mimic the formation of a complex 3D environment. Currently they are used in regenerative medicine, preclinical in vitro testing and to engineer vascular, neural, bone, cartilage, ligament, skin, and skeletal muscle tissues.
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| A) Porous Disc (B) Electrospun and (C) Orthogonal layering; geometric configurations of polymeric 3D scaffolds. Image Source: Haycock, J.W. (2011) |
b. Biological Scaffolds
Scaffolds created from natural or biological substances such as commonly found proteins in the ECM. Eg: Include fibronectin, collagen, laminin, and gelatin proteins.
They are unique in sense that they supply the right microenvironment of soluble growth factors, hormones and molecules interacting with cells in vivo which can modify gene & protein expression. Current methods include mixing of cells with scaffold proteins in a liquid condition (hydrogel formation), or superficial application of protein mixture onto cells already aggregated into 3D spheroids.
c. Micropatterned Surface Microplates
These contain plates with micrometer sized compartments, arranged regularly at the bottom of wells. The wells could be either squared, round, or squared with slits between the walls of adjacent wells. The plate to be chosen depends on the cell type used and the networking formation or spheroid formation.
2. Non-Scaffold Based
a. Hanging Drop MicroPlates (HDP)
These enable the aggregation of the cells into a 3D spheroid due to the absence of a surface for attachment. The bottom of the wells is such that a small drop of surface media subtends into it and over a period of time, a mass of cells forms due to surface tension.
These are best used for in vivo-like examination of tumor metastasis.
b. Spheroid Microplates containing Ultra-Low Attachment (ULA) coating
These are plates with a round, tapered or V-shaped geometry to allow for the creation of a single, consistent spheroid. The ULA coating basically minimizes cell adherence to increase efficiency of spheroid formation.
Microfluidic 3D Cell Culture
These impart an additional complexity to the model by introducing small capillaries or microchannels which could be used to manipulate the fluidic environment at physiological conditions. These mimic the vasculature around cells in vivo which maintains a continuous nourishing supply of gases, enabling the creation of a closer biomimetic model. Often natural polymers like collagen, fibrin or agarose are used to create such microfluidic devices which are used for tissue engineering.
Applications of Microfluidics include creation of an:
− organ-on-a-chip technology: a microfluidic perfusion device for regenerative medicine.
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| 1. An organ-on-a-chip showing wells; tissues / spheroids are maintained in these wells. (Picture Credits: Kbjung, Wikimedia Commons License Details, The picture was cropped to obtain a better view.). |
− chip-based platform: for the culture of cells and toxicological studies |
| 2. A microfluidic plate showing perfusion of a liquid into the culture. Source: (Microfluidic Technologies) |
It involves printing of living cells, layer‐by‐layer into special 3‐D scaffolds. Initially, the bioprinters used were modified versions of the 2D ink-based printers, in which the ink was replaced with a biological material whereas the paper was replaced with a stage with controlled elevator control of the 3 Axes.
2. An organ-on-a-chip showing wells; tissues / spheroids are maintained in these wells. (Sour
However, recent bioprinters utilize thermal or acoustic means to eject drops of bioink (tissuce: Microfluidics)es) onto substrate. Bioiprinting materials are of natural origin (Eg: alginate, collagen, chitosan, fibrin) and cell lines are used as bioink due to their robust nature and ability to survive mechanical stress.
They have a huge potential in regenerative medicine to construct complex functional tissues and organs, and more recently it has served to generate blood vessels needed for vasculature8. Such artificially constructed tissues could also be utilized in drug discovery, toxicological & chemical analyses and cancer research.
HLA Typing
Human leukocyte antigen (HLA)-typing enables the transfer of plasma without any graft rejection issues, hence this is of immense importance in immunotherapy and organ transplantation. Since generating HLA typing results for specific cells of body is time consuming, a library of HLA-typed cells specific for each different type of body tissue is maintained.
Conclusion: The Future of Cell Culturing
As the emphasis on 3D Cell culturing is increasing with each recurring day, we would have increased applications in Tissue Engineering, Transplantational studies. Moreover, ever since the production of recombinant somatostain, many more therapeutic proteins have been produced. The future would probably also see the usage of stem cells (adult as well as embryonic) for the replacement of damaged tissues and surgical reconstruction.
References
1. Cover Page Image from Stem Cell Technologies: CHO Cells and Hybridomas. Available from: https://www.stemcell.com/tissue-and-cell-types/cho-cells-and-hybridomas.html(Accessed on 9thMarch, 2021)
2. Animal Cell Culture – Qiagen Website.
3. Cell Culture: Basics, Techniques and Media – MicroscopeMaster Website.
4. Introduction to Cell Culture – ThermoFisher SCIENTIFIC.
5. Jedrzejczak-Silicka, M. (2017). History of cell culture. New insights into cell culture technology. DOI: 10.5772/66905.
6. Verma, A. (2014). Animal tissue culture: Principles and applications. In Animal Biotechnology(pp. 211-231). Academic Press.
7. Souza, A. G., Ferreira, I. C. C., Marangoni, K., Bastos, V. A. F., & Goulart, V. A. (2016). Advances in cell culture: more than a century after cultivating cells. Journal of Biotechnology & Biomaterials, 6(2).
8. Animal Cell Culture Trends in Biomedical Research – Kosheeka Website. Available from: https://kosheeka.com/animal-cell-culture-trends-in-biomedical-research/ (Accessed on 10thMarch, 2021)
9. Larson B., (2015). 3D Cell Culture: A Review of Current Techniques. BioTek Resources - White Papers (Accessed on 10th March, 2021)
10. Haycock, J. W. (2011). 3D cell culture: a review of current approaches and techniques. 3D cell culture, 1-15.
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