The Importance of Tissue Dissociation Enzymes in Cell & Tissue Culture
Cells are the basic functional unit of life. Cells of specialized type come together to form a tissue, whereas tissues that perform a specific function compose an organ. Cell culture plays a significant role in studying basic cell biology, cell cycle mechanisms, specialized cell function, cell–cell and cell–matrix interaction. Tissue culture allows for study of cell biology from multicellular organisms in vitro either as 2-D or 3-D cell cultures. Today, advancements in scientific methodology and cell culture matrix reagents provide in vitro environments conducive for 3-D cell culture, where stem cells can be differentiated and grown into organoids, that mimic the cell arrangement of an organ. This blog focuses on the technique of cell culture, 2-D and 3-D, and the importance of tissue dissociation enzymes for cell separation. Cell Culture vs Tissue Culture: What’s the Difference? Tissue culture and cell culture are often used interchangeably. So one may wonder, what is the main difference? The source of cells propagated in vitro. Cell culture is the artificial maintenance of dissociated cells in vitro, either isolated as primary cells, immortalized cell lines or cell strains. This in vitro environment requires supplemental media and growth factors to maintain cell growth. Cell culture studies complement in vivo experiments, as the cells allow for a more controlled environment to assess cellular functions and processes. Tissue culture is when cells are removed from a parent organism and viable propagation of isolated cells, tissues or organs is maintained in vitro through artificial means. Tissue culture allows for study of cell biology from multicellular organisms. Tissue culture experiments often assess physiological function of cells that make up the tissue and can be used for pharmacological assessment (e.g. cytotoxicology) before moving to in vivo experiments. What Are Some Common Uses of Cell & Tissue Culture? Scientists can perform various down-stream assessments from cell culture experiments to decipher mechanisms of action, post-translational modifications and cell-cell relationships. To do these studies, antibodies and cell markers in applications like western blot or flow cytometry can be used to determine protein expression or localization. Furthermore, single-cell applications like immunocytochemistry can also provide the localization of specific proteins in or on a cell, whereas scRNA-seq provides genomic characterization. These types of analyses can help one understand the relationship of regulatory genes and protein expression of a cell. This provides an avenue for understanding cell function when experimental modifications in vitro can be directly correlated with protein or genetic alterations observed at the single-cell level. Regenerative medicine using pluripotent stem cells use 3-D cell culture experimental techniques to develop organoids. Organoids derived from stem cells aggregate together to form a spheroid structure that mimics an in vivo organ. To obtain this 3-D structure, extracellular matrices help form the cellular bonds & organization of cells in vitro. Organoids can be used to study human development and disease, drug discovery and perhaps personalized medicine. What Is Tissue Dissociation? Tissue dissociation can employ mechanical dissociation, enzymatic dissociation or a mixture of both methods to obtain primary cells from tissue. In short, mechanical dissociation is when a tissue sample is cut, crushed, or pulverized/homogenized using an instrument to get the tissue in smaller, digestible pieces. Mechanical dissociation is often required for tissues that have strong adhesions that need to be broken down before single cells can be extracted. Tissues that undergo harsh mechanical dissociation may result in low viability and low yield of primary cells. A less harsh method of dissociation is mechanical disruption, in which using a pipette (for trituration) or a vortex can help disaggregate the cells from softer tissue with less physical force, improving viability and yield. Enzyme dissociation, or enzymatic digestion, used in tissue dissociation protocols is important for cell harvesting of primary cells and can often result in the same extraction efficiency as mechanical dissociation. One important factor when assessing your experimental design for enzymatic dissociation is that temperature plays a significant role in enzyme activity. The stability of individual enzymes varies as do their optimal temperature-dependent behavior–where their enzymatic activity is greatest for tissue digestion. Therefore, each experimental protocol requiring tissue dissociation should be optimized based on the tissue type and sensitivity whether it uses mechanical dissociation, enzymatic digestion, or both, to provide optimal primary cell isolation for cell culture. Specific enzymes used in various tissue digestion processes for cell isolation will be discussed in sections below. What Are Primary Cells? Primary cells are directly isolated from fresh tissue of a living organism. Primary cells can be obtained from tissue biopsies or from organ removal. Primary cells are more sensitive than cell lines and require more supplements than classical media. Primary cells are grown and maintained in vitro using various cell culture reagents such as fetal bovine serum, classical medium, supplements, extracellular matrices and antibiotics to minimize contamination growth. Primary cells are not passaged like cell lines and have a finite lifespan in vitro. What Are The Differences Between Primary Cell Culture and Immortalized Cell Lines (Secondary Cell Culture)? Primary cells have a limited life span in vitro whereas secondary cells (immortalized cell lines) have an unlimited lifespan in culture with the right conditions. Cell lines are derived from an existing primary cell culture and are created to expand the lifespan and proliferation rate of that cell type. In fact, a main reason cell lines are often utilized in cell culture experiments is that cell lines can be maintained and passaged, providing a consistent experimental cell model. Secondary cell cultures also tend to be more robust, being less sensitive to in vitro changes than primary cells. Cell lines are also easier to transfect when performing gene engineering experiments, allowing for higher viability after transfection in cell lines than in sensitive primary cells. This experimental flexibility is a benefit for scientists who are investigating specific questions about cellular biology or function at the cellular level for a specific cell type. However, primary cell cultures are better representations of functional components of tissue compared to immortalized cells as they are a closer match to an