Primary cell culture | |
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Primary interstinal organoid culture | |
Identifiers | |
MeSH | D061251 |
Anatomical terminology |
Primary cell culture is the ex vivo culture of cells freshly obtained from a multicellular organism, as opposed to the culture of immortalized cell lines. In general, primary cell cultures are considered more representative of in vivo tissues than cell lines, and this is recognized legally in some countries such as the UK (Human Tissue Act 2004). [1] However, primary cells require adequate substrate and nutrient conditions to thrive and after a certain number of divisions they acquire a senescent phenotype, leading to irreversible cell cycle arrest. [2] The generation of cell lines stems from these two reasons. Primary cells can become immortalized either spontaneously (e.g. HeLa cells) or by genetic modification (e.g. HEK cells), at which point they become cell lines which can be subcultured indefinitely. [3]
Because of their requirements for viability, primary cell cultures did not become widespread until the 2000s. These cultures present several advantages over cell lines, including a better representation of the cellular heterogeneity of tissues, a more faithful transcriptomic and proteomic profile (especially when cultured in 3D) and more realistic functional responses, including drug responses. [4] [5] [6] In contrast, immortalized cell lines are known to become homogeneous through the natural selection of specific subpopulations, to undergo genetic drift and to acquire genetic aberrations. In many cases, cell lines have been misidentified, contaminated with other cells or infected with Mycoplasma , small intracellular bacteria that went undetected for decades. [4] [7]
When whole or partial tissues are isolated and maintained ex vivo, the procedure is termed primary tissue culture . More specific terms include organotypic culture, [8] tissue slices [9] and explants. [10]
Neuronal primary cell cultures are cells collected from the brain of an organism. For example they can be used when examining substances effect on cell viability, which can further on be potential treatments for brain deficits. [11]
"Monolayer cultures" refer to cell cultures where cells are grown in a single, flat layer on the surface of a culture dish or substrate. In a monolayer culture, cells adhere to the substrate and spread out in a two-dimensional arrangement. This type of cell culture is commonly used in laboratory settings for various purposes, including research, drug testing, and biotechnology.
Key features of monolayer cultures include:
Two-Dimensional Growth:Cells in monolayer cultures grow in a single plane, adhering to the surface of the culture vessel. This flat arrangement allows for easy observation and manipulation of individual cells. [12]
Adherence to Substrate:The cells attach to the surface of the culture dish or flask, and their growth and behavior can be influenced by the characteristics of the substrate.on the other words In cell culture, adherence to substrate describes a cell's capacity to adhere to a surface and proliferate.Many elements, including surface energy, substrate topography, and roughness, mediate the process of cell attachment. [13] The study of artificial polymer surfaces with varying chemical, topological, and mechanical cues that regulate cell activities has focused attention on the interaction between external surfaces and cells. [13] In a study that was published in the journal RSC Advances in 2021, the impact of roughness and surface energy on cell adhesion and growth was examined.The most advantageous circumstances for effective cell adhesion, development, and proliferation were discovered by the study to be moderate surface energy and intermediate roughness ratio. [13]
Cell Proliferation:Cells in monolayer cultures can undergo cell division and proliferation. This feature is crucial for experimental studies and the production of a larger number of cells for subsequent analyses. [12]
Observation and Imaging:The two-dimensional nature of monolayer cultures makes it convenient for microscopic observation and imaging. Researchers can easily visualize the cells, study their morphology, and monitor changes over time. [12]
Cell Differentiation:Depending on the cell type and culture conditions, monolayer cultures can be used to induce cell differentiation. This is particularly important in studying developmental processes and tissue-specific functions. [12]
Endocrine cancer with the highest incidence is thyroid cancer (TC). Differentiated thyroid cells (DTC) that originate from follicular thyroid cells account for over 90% of total thyroid cells (TC). Papillary TC (PTC), follicular TC (FTC), and Hürthle cell TC are examples of DTC. One percent of TC is anaplastic TC (ATC), which accounts for 15–40% of TC deaths. [14]
Mortality is one of the biggest obstacles to current treatment techniques against aggressive DTC or ATC. These strategies are not entirely effective against these conditions. Recent years have seen advancements in our knowledge of the molecular and genetic underpinnings of TC development as well as the introduction of novel medications, such as tyrosine kinase inhibitors (TKIs), which target the oncogenic or signaling kinases linked to cellular proliferation. [14]
Preclinical models have made use of thyroid cell lines that were isolated from tumoral cells and selected for their high rate of proliferation in vitro. As a result of their adaptation to in vitro growth circumstances, these cells actually lose the distinctive characteristics of the original tumor. Because of these factors, there are significant restrictions on the usage of these cell lines. More recently, monolayer cultures of human primary cells have been created, and their biological behavior has been studied. Furthermore, whereas human primary cell cultures may now be created from samples of fine-needle aspiration citology from aggressive dedifferentiated DTC or ATC, primary TC cells were previously only obtained by surgical biopsies. Without the use of useless medications, testing several TKIs in vitro on individual patients can aid in the development of novel, individualized treatments. [14]
Limitations of monolayer culture and motivations:
Scientists are investigating novel models that can more accurately mimic the structure and function of human organs because to the limitations of monolayer culture settings. Protocol improvements in recent times have led to the creation of three-dimensional (3D) organ-like architectures known as "organoids," which are able to exhibit properties of their corresponding real organs, such as morphological features, functional activities, and individual responses to particular pathogens. [15]
For in vitro investigations to be conducted correctly, the cell culture protocol for a particular cell line must be optimized. The best culture conditions for different cell lines can differ significantly due to the heterogeneity of germ cell malignancies. [16]
A fibroblast is a type of biological cell that synthesizes the extracellular matrix and collagen, produces the structural framework (stroma) for animal tissues, and plays a critical role in wound healing. Fibroblasts are the most common cells of connective tissue in animals.
Thyroid neoplasm is a neoplasm or tumor of the thyroid. It can be a benign tumor such as thyroid adenoma, or it can be a malignant neoplasm, such as papillary, follicular, medullary or anaplastic thyroid cancer. Most patients are 25 to 65 years of age when first diagnosed; women are more affected than men. The estimated number of new cases of thyroid cancer in the United States in 2010 is 44,670 compared to only 1,690 deaths. Of all thyroid nodules discovered, only about 5 percent are cancerous, and under 3 percent of those result in fatalities.
Metastasis is a pathogenic agent's spread from an initial or primary site to a different or secondary site within the host's body; the term is typically used when referring to metastasis by a cancerous tumor. The newly pathological sites, then, are metastases (mets). It is generally distinguished from cancer invasion, which is the direct extension and penetration by cancer cells into neighboring tissues.
An ovarian follicle is a roughly spheroid cellular aggregation set found in the ovaries. It secretes hormones that influence stages of the menstrual cycle. At the time of puberty, women have approximately 200,000 to 300,000 follicles, each with the potential to release an egg cell (ovum) at ovulation for fertilization. These eggs are developed once every menstrual cycle with around 450–500 being ovulated during a woman's reproductive lifetime.
Cell culture or tissue culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. The term "tissue culture" was coined by American pathologist Montrose Thomas Burrows. This technique is also called micropropagation. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. They need to be kept at body temperature (37 °C) in an incubator. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or rich medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar. Tissue culture commonly refers to the culture of animal cells and tissues, with the more specific term plant tissue culture being used for plants. The lifespan of most cells is genetically determined, but some cell-culturing cells have been “transformed” into immortal cells which will reproduce indefinitely if the optimal conditions are provided.
Matrigel is the trade name for the solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced by Corning Life Sciences. Matrigel resembles the laminin/collagen IV-rich basement membrane extracellular environment found in many tissues and is used by cell biologists as a substrate for culturing cells.
3T3 cells are several cell lines of mouse embryonic fibroblasts. The original 3T3 cell line was established in 1962 by two scientists then at the Department of Pathology in the New York University School of Medicine, George Todaro and Howard Green. Todaro and Green originally obtained their 3T3 cells from Swiss albino mouse embryo tissue. Later, as a principal investigator position at the National Cancer Institute in Bethesda, Maryland, Todaro repeated the isolation procedure from the NIH Swiss mouse embryo with his students and established NIH-3T3 cell line.
An organoid is a miniaturised and simplified version of an organ produced in vitro in three dimensions that mimics the key functional, structural and biological complexity of that organ. They are derived from one or a few cells from a tissue, embryonic stem cells or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities. The technique for growing organoids has rapidly improved since the early 2010s, and The Scientist names it as one of the biggest scientific advancements of 2013. Scientists and engineers use organoids to study development and disease in the laboratory, drug discovery and development in industry, personalized diagnostics and medicine, gene and cell therapies, tissue engineering and regenerative medicine.
Papillary thyroid cancer is the most common type of thyroid cancer, representing 75 percent to 85 percent of all thyroid cancer cases. It occurs more frequently in women and presents in the 20–55 year age group. It is also the predominant cancer type in children with thyroid cancer, and in patients with thyroid cancer who have had previous radiation to the head and neck. It is often well-differentiated, slow-growing, and localized, although it can metastasize.
SH-SY5Y is a human derived cell line used in scientific research. The original cell line, called SK-N-SH, from which it was subcloned was isolated from a bone marrow biopsy taken from a four-year-old female with neuroblastoma. SH-SY5Y cells are often used as in vitro models of neuronal function and differentiation. They are adrenergic in phenotype but also express dopaminergic markers and, as such, have been used to study Parkinson's disease, neurogenesis, and other characteristics of brain cells.
T-cadherin, also known as cadherin 13, H-cadherin (heart), and CDH13, is a unique member of the cadherin superfamily of proteins because it lacks the transmembrane and cytoplasmic domains common to all other cadherins and is instead anchored to the cell's plasma membrane by the GPI anchor.
Mucin-4 (MUC-4) is a mucin protein that in humans is encoded by the MUC4 gene. Like other mucins, MUC-4 is a high-molecular weight glycoprotein.
Paired box gene 8, also known as PAX8, is a protein which in humans is encoded by the PAX8 gene.
Follicular thyroid cancer accounts for 15% of thyroid cancer and occurs more commonly in women over 50 years of age. Thyroglobulin (Tg) can be used as a tumor marker for well-differentiated follicular thyroid cancer. Thyroid follicular cells are the thyroid cells responsible for the production and secretion of thyroid hormones.
Arginylglycylaspartic acid (RGD) is the most common peptide motif responsible for cell adhesion to the extracellular matrix (ECM), found in species ranging from Drosophila to humans. Cell adhesion proteins called integrins recognize and bind to this sequence, which is found within many matrix proteins, including fibronectin, fibrinogen, vitronectin, osteopontin, and several other adhesive extracellular matrix proteins. The discovery of RGD and elucidation of how RGD binds to integrins has led to the development of a number of drugs and diagnostics, while the peptide itself is used ubiquitously in bioengineering. Depending on the application and the integrin targeted, RGD can be chemically modified or replaced by a similar peptide which promotes cell adhesion.
A 3D cell culture is an artificially created environment in which biological cells are permitted to grow or interact with their surroundings in all three dimensions. Unlike 2D environments, a 3D cell culture allows cells in vitro to grow in all directions, similar to how they would in vivo. These three-dimensional cultures are usually grown in bioreactors, small capsules in which the cells can grow into spheroids, or 3D cell colonies. Approximately 300 spheroids are usually cultured per bioreactor.
Noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) is an indolent thyroid tumor that was previously classified as an encapsulated follicular variant of papillary thyroid carcinoma, necessitating a new classification as it was recognized that encapsulated tumors without invasion have an indolent behavior, and may be over-treated if classified as a type of cancer.
Horizontal transfer of mitochondria is the movement of whole mitochondria and mitochondrial DNA between cells. Mitochondria from donor cells are transported and incorporated into the endogenous mitochondrial network of recipient cells contributing to changes in the bioenergetics profile and in other functional properties of recipient cells. Horizontal cell-to-cell transfer of mitochondria and mitochondrial genome can occur among mammalian cells in vitro and in vivo. Mitochondrial transfer supports the exogenous replacement of damaged mitochondria, thereby rescuing mitochondrial defects. Stem cells, immortalized cells or primary cells are usually used as mitochondrial donors in most studies. These cells may transfer mitochondria to surrounding cells in their niche, thus affecting cell differentiation, proliferation, tissue homeostasis, development and ageing.
Cultrex Basement Membrane Extract (BME) is the trade name for a extracellular protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and manufactured into a hydrogel by R&D Systems, a brand of Bio-Techne. Similar to Matrigel, this hydrogel is a natural extracellular matrix that mimics the complex extracellular environment within complex tissues. It is used as a general cell culture substrate across a wide variety of research applications.
MIR22HG, also known as C17orf91, MGC14376, MIRN22, hsa-mir-22, and miR-22 is a human gene that encodes a noncoding RNA (ncRNA).This RNA molecule is not translated into a protein but nonetheless may have important functions.
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