| Abstract|| |
Cell culture is one of the most valuable tools which is being applied in both fundamental and applied gastrointestinal research. The cells are isolated from their natural location (in vivo) and further propagated in vitro or artificial environment and studied. Over the years, several methods have been devised to isolate animal cells derived from the gut and culture them in vitro to study the functions and biology in the context of complex gastrointestinal diseases. This mini-review briefly describes the types and methods of cell culture covering the simplest monoculture models to more recent 3D organoid models, highlighting its importance in personalized precession medicine and other aspects of translational research. It also throws light upon the major challenges and outlines the future directions for using cell culture as a model system.
Keywords: 2D culture, cell lines, co-culture, contamination, organ on chip, organotypic cultures
|How to cite this article:|
Preksha G, Yesheswini R, Srikanth CV. Cell culture techniques in gastrointestinal research: Methods, possibilities and challenges. Indian J Pathol Microbiol 2021;64, Suppl S1:52-7
|How to cite this URL:|
Preksha G, Yesheswini R, Srikanth CV. Cell culture techniques in gastrointestinal research: Methods, possibilities and challenges. Indian J Pathol Microbiol [serial online] 2021 [cited 2021 Jun 23];64, Suppl S1:52-7. Available from: https://www.ijpmonline.org/text.asp?2021/64/5/52/317932
| Introduction|| |
Mammalian cell culture systems are very flexible and resourceful in vitro tools, which have widely aided basic and applied biomedical research. These model systems are easy to handle, genetically tractable and closest to in vivo conditions. These models are preferred in cancer biology, drug discovery, tissue engineering, pharmaceutical research, vaccine development, genetic engineering, and several other domains of life science [Figure 1]. The cells can be directly isolated as “primary cells” from donor or cell banks, such as American Type Culture Collection (ATCC), a repository of both primary cells and cell lines. Depending upon the research problem, cells and cell lines with required cultural conditions have to be chosen. Taking proper care of personal safety and sterility of the working before and after the work is very important, as there are always chances of exposure to biohazard and contamination of cell culture. Appropriate containment levels (CL 1, 2, and 3) have been devised based on the assessment of biological risks while working with tissue culture using specific biosafety cabinets (BSC 1, 2, and 3) to provide protection to the user [Figure 2]. These biosafety cabinets should be compliant to the safety guidelines (having HEPA/ULPA filters) and proper airflow with acceptable quality certification (e.g., EN certified). Some of these aspects are covered in WHO biosafety manual. Also, the protocols for using cell culture has to be approved by the Institutional Biosafety Committee (IBSC) and Review Committee on Genetic Manipulation (RGCM) committee by the user.
|Figure 2: Diagram showing the containment levels to adopt while working in cell culture|
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In the last 4 − 5 decades significant progress has been made in the field of cell culture, leading to remarkable breakthroughs in basic and applied fields. From two dimensional (2D) monolayer culturing, the field has surged to a more sophisticated technology of organotypic culturing or three-dimensional (3D) culturing. Cell culture is a most opted model system for understanding of complex organ systems, and thereby newer ideas and innovations emerge rapidly in this field. These systems are compatible with state-of-the-art tools of molecular biology and genetic engineering and therefore have noteworthy adaptability for various fields including Gastrointestinal (GI) research. As reviewed by Dutton et al. and Costa et al.,, the GI tract is immensely vast with several organs and functions well researched using cell culture systems. The current review intends to cover cell culture model systems that are commonly used in GI field. Intestinal epithelial cell culture tools will be described involving primary culture and 2D cultures, co-culture methodologies followed by the more sophisticated 3D culturing methods that are fast emerging in the field. This work will be particularly useful for students and beginners who would like to initiate work in GI research using cell culture systems.
Simple cell culture systems and applications
Primary intestinal cells are obtained directly from tissue (small or large intestine) in case of small animal models, from cadavers, surgically resected specimens from live subjects, or through colonoscopy. The cells are harvested by subjecting the tissue with chemical, enzymatic, or mechanical digestion to get rid of the extracellular matrix (ECM). After isolation, they are diluted in suitable commercial growth media and propagated in sterile culture vessels in a cell culture facility [Table 1]. Direct culturing from tissue bestows features closest to the parent organ. Being from primary origin, they are not immortalized and have a definitive lifespan and therefore need to be frequently cryopreserved. Though there are several examples of culturing murine cells (ATCC) and human cells (LONZA), working with primary cells can be challenging, as several of them fail to adhere and proliferate in vitro and are not amenable to molecular techniques.
In cases where the feasibility of primary cells is challenging, researchers utilize immortalized or cancerous cells cultured as anchored “Two-dimensional” lines. Examples of some of these intestinal derived lines, which are relatively easier to grow, are given in [Table 2]. Cells are grown as a monolayer as they attach to the flask bottom surface. Under adequate nutritional and growth conditions [Table 3] they proliferate until becoming confluent, covering all available surface area in the culture-flask. Confluent cells are to be sub-cultured in order to overcome nutrient deprivation and death. Monolayer cell culture has a characteristic growth kinetics: lag phase (initial no growth period after seeding), log phase (exponential growth), and stationary phase (constant cell number due to nutrient deprivation and cell-to-cell contact inhibition). The major limitation of monolayer cell culture is that it does not focus upon the co-ordinate interplay between different cell types that could actually mimic in vivo microenvironment as 2D culture consists of single cell type. These cell types have cell morphology, polarity, receptor expression, oncogene expression, cell-ECM interaction which may not be necessarily similar to those from cells in vivo. A variant and non-adherent culture type is Suspension Culture, in which cells are independent of surface anchorage. The cells proliferate even under floating conditions. Advantage of this method is it allows us to study metabolic interaction and production of metabolites in chemostat or turbidostatic steady-state system.
|Table 2: List of instestinal derived cell lines and their use in different in vitro culturing systems|
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A range of different assays to test critical cell and tissue physiology is doable with using above mentioned simple forms of cell culture. These include testing several of the important capacities of the cells such as their ability in proliferation migration, secretion, resist infection and injuries etc. Some of these are described below.
Wound healing assays are standard 2D culture-based assays, used to gauge capacity of cell migration, a cell-free area is created by physical or mechanical exclusion of cells. The capacity of cells to migrate and proliferate is then monitored. Cancerous cells are also gauged by a more sophisticated technique called Transwell assay using Boyden chambers, a porous membrane on a cylindrical insert in the culture well. In this way the cells are grown and their ability to migrate across the membrane is monitored. Growing epithelial cells such as human colon carcinoma cell line T84 in Transwell format in which they display key intestinal epithelial markers including tall columnar appearance, polarization into apical, and basolateral surfaces, expression of tight junction proteins (which also help in barrier function), and also the development of transepithelial electrical resistance (TEER) which is a feature acquired due to epithelial barrier function. TEER can be measured using a voltmeter specifically designed for this purpose. The same Transwell setup may be used to study epithelial cells and immunocytes crosstalk by co-culturing epithelial cells in the Transwell and immune cells in the culture well [Figure 3]. A combination of experimentally perturbed murine epithelial cells cocultured with total immunocytes of mice helped understanding of a unique mode of epithelial immunocyte crosstalk involving a specific category of T cell that was crucial in pathogenesis of inflammatory bowel disease. Such assay is called as polymorphomononuclear leucocyte assay or PML assay and have been extensively used to understand Salmonella Typhimurium induced gut inflammation., Similarly this can be combined with a microbial infection as is done in transcytosis assays. Soft agar culturing to monitor abilities of cells to form colonies is utilized to gauge anchorage dependence of cells in cancer biology.
|Figure 3: Types of 2D and 3D culture models used in gastrointestinal system study. (Created with BioRender)|
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Most of these models are amenable to genetic and molecular biology manipulations, including knock-ins, knock-downs, and knock-outs using CRISPR editing tools. Gene specific reagents for RNA interference (RNAi) for cell culture are readily available. These may be used along with control RNAs and gene-specific transient knock-downs can be studied. Vectors that enable stable expression of heterologous constructs have been used for knock-ins and conditional expression. Transfection is done by either using gene transfection reagent or electroporation, and subsequently, the clones of cells expressing transgene are isolated and expanded in appropriate selection media. For genomic editing lentiviral transfection could be done using CRISPR-CAS9 system.
While these tools involving monolayer and suspension cultures have enabled amassing huge amount of information facilitating mechanistic studies. But somehow they have failed in drug discovery programs and due to the limited translatability of the massed data. This may be like due, at least in part, to the insufficient representation of tissue properties including immune cells, and inadequate heterogeneity including lack of stroma and extracellular matrix (ECM). Due to these weaknesses, more sophisticated 3D cultures have emerged and their popularity is growing rapidly.
As described above, complex human diseases that involve participation of multiple cell types cannot be understood using regular 2D culture systems. For a better understanding of relevance and to overcome the shortcomings of 2D cell culture, three-dimensional (3D) cell culture technique has been developed. In addition to being three dimensional they also closely mimic tissue microenvironment and cell-cell dynamics [Table 4]. Organoids represent more than one cell type and recapitulate the architecture and gene expression from its parent organ. Because of these salient features organoid systems have been known to mirror in vivo environment very well. Types of 3D culture can be broadly classified into scaffold-free and scaffold based [Figure 4]. Scaffold-free type is preferred by spheroids and scaffold based type is preferred by cells or tissues that require ECM anchorage which can be attained either by seeding cells in 3D ECM matrix or by dispersion of cells in liquid hydrogel later allowing it to polymerize. This breakthrough could be possible due to in-depth understanding of molecular signaling that govern intestinal stem cell (ISC) behavior, making it possible for long term expansion of Lrg5 expressing ISCs into structures called as 'mini-gut'. Organoids have a central hollow, several protruding structures, Lrg5 positive cells and Paneth cells, thus resembling the crypts of small intestine. Tissue-specific adaptation has enabled derivation of organoids from almost all organs of gastrointestinal tract, including small intestine, colon, stomach, gallbladder, esophagus, liver, pancreas, salivary glands and even taste buds. Organoids are amenable to genetic manipulations and can also be cocultured with microbes to understand pathogenesis and host-microbe interactions., For instance, CRISPR-CAS9 gene editing tools were used by Drost et al. to introduce mutations in cancer related genes (KRAS, APC, SMAD and TP53) in cultured organoids derived from human intestinal tissue of colorectal cancer patients. Organoids having all the four mutations (engineering organoids) overcame the requirement of niche factors such as R-spondin, WNT3A and epithelial growth factors. These engineered organoids when xenografted into immunodeficient mice by subcutaneous transplantation showed development of aggressive metastatic tumors that were highly proliferative, invasive with several other cellular and molecular features. Similarly the importance of microbiota and CRC was investigated using organoids. Selected microorganisms when microinjected into cultured intestinal organoids exacerbated tumor development. Such studies involving organoids have given critical insights in genes and regulators that govern progression of gut illnesses. In addition, multiple investigations involving collection of organoids derived from patients representing different categories of the diseases have established in the forms of 'organoid biobanks'. These organoids from colonic and other gut tissues have been bio banked and extensively examined for molecular and transcriptomic signatures. First and foremost it was seen that these organoids mimicked exactly the same genomic and transcriptomic profile as the parent tissue. Several subcultures of the organoids were then established and used for screening of drugs. This is a very interesting and promising arena since it has potential in translational research and therapeutics. As highlighted above, specifically in cancer research these have been engaged as an ex vivo model for disease modeling, regenerative medicine, therapeutic research and drug discovery. They have also been bio banked to enable research in these areas retrospectively for developing personalized medicine.
|Figure 4: Different types of methods and innovations in 3D in vitro culturing. (Created with BioRender)|
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Organs-on-Chip Next wave of 3D culture model
As discussed in previous sections, 3D culture system is a major headway in biomedical and translational research, over 2D culture, by providing higher levels of tissue organizations. Yet, they fail to mimic an organ's cellular properties in several regards such as tissue-tissue interfaces and mechanical microenvironments. Intestine as a part of gastrointestinal system contributes to several pathological disorders such as inflammatory bowel disease, colorectal cancer, radiation enteropathy and diabetes etc. To maintain human health, it is required to establish and preserve a balanced intestinal interaction and gut homeostasis.
The application of microfluidics in organs-on-chips seems to be a promising innovation permitting the study of human physiology in an organ specific context. An organ-on-chip (OOC) is a 3D microfluidic cell culture chip [Figure 4] that inhabits the 3D microarchitecture and environment of organ system exhibiting cell-cell, cell-microbe and other interactions. Intestine-on-chip consists of a glass slide permeable membrane and PDSM sheet with channels. Intestinal epithelial cells (Caco2) are cultured over these chips alone or with endothelial cells (HUVECs) showing prolonged growth and maintained microbial flora. These chips have opened entirely new possibilities to study polarized epithelial cells and their interactions with immune system, vascular networks and microbiome. Individual variations can also be replicated over these small chips providing better ways for development for personalized medicine. Although OOC technology has developed rapidly and demands sophisticated and expensive manufacturing and implementations, this can be an alternative to overcome the shortcomings of 2D cell culture and complications in establishing 3D organoid models.
Cell culture is most widely employed system in research field, one of the main limitations of this model system is expense and effort. From the basic setup to highly sophisticated advance techniques in cell culture are pricey. Also, it requires a highly skilled personnel and techniques are to be performed in highly aseptic environment. While handling mammalian cell culture, contamination is a major challenge. These can be either biological contamination or cross contamination. Biological contamination mostly occurs due to microorganisms like bacteria (~2 μm), yeast (~4 μm), viruses (~300 nm) or mycoplasma (<1 um). Cross contamination mainly involves growth of more than one cell type in a culture flask or experiment. Any type of contamination can severely affect the cells in culture genetically and morphologically and may produce unacceptable results.
Future perspectives of cell culture
Since use of cell culture is versatile, its applications can be widened through bringing up innovations. Improvements in microfluidics-based organ on chip system can be used to study drug development, disease remodelling and personalized medicine. Development in technologies for generation of mammalian cell lines for production of recombinant proteins for therapeutics are vital in the field of biopharmaceutical industry. Creation of such a cell line that can meet the goal of synthesizing quantitatively and qualitatively effective recombination protein is labour intensive and time-consuming process. But generation of such cell lines can pave a way towards drug discovery and development.
| Conclusions|| |
Cell culture has been a prominent field of biosciences with usage in aspects of biological research. But now this has gone beyond just conventional investigational procedures to technology related applications such as gene therapy, pharmacology, stem cell biology etc. This review summarizes the basic cell culture methods and required components particularly used in studies related to gastrointestinal system. Despite few challenges faced and expenses, it has proven to be a promising model system to study and understand cellular functions and other biological characteristics over in vivo systems. Cell culture has come with lots of important applications in medical advancements and has ultimately enabled for the development of breakthrough clinical uses.
Work in our lab is supported by Scheme for Transformational and Advanced Research in Sciences-Ministry of Human Resource Development (STARS-MHRD) grant and core grant from Regional Centre for Biotechnology (RCB).
Financial support and sponsorship
Work in our lab is supported by STARS-MHRD grant of CVS and core grant from RCB.
Conflicts of interest
There are no conflicts of interest.
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Chittur V Srikanth
Regional Centre for Biotechnology, 3rd Milestone, Faridabad-Gurugram Expressway, Faridabad - 121 001, Haryana
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]