Cell Culture Techniques Flashcards
What is cell/tissue culture?
Lab method by which cells are grown under controlled conditions outside their natural environment
What are the advantages of cell culture techniques?
Control of the physiochemical environment and physiological conditions
Control of the micro-environment of the cells
Cells can be easily characterised by cytological and immune staining techniques and visualised using imaging techniques
Cells can be stored in liquid nitrogen for long periods
Cells can be easily quantified
Reduces use of animals in scientific experiments
Cheaper to maintain
What is the physiochemical environment?
pH, temperature, osmolarity
What are the physiological conditions?
Hormone and nutrient levels
What is the microenvironment of the cells?
Matrix, cell-cell interactions and cell substrate attachment
What is cryptopreservation?
Cells can be stored in liquid nitrogen for long periods
What are the disadvantages of cell/tissue culture?
Inter-patient variation Limited number (at high cost) Finite lifespan and hard to maintain Difficult molecular manipulation Phenotypic instability Variable contamination
What are the methods of isolation?
Cells allowed to migrate out of an explant
Mechanical dissociation
Enzymatic dissocitation
What are the characteristics of primary tissue cells?
Cells derived directly from tissues/ patients
Finite lifespan
Cells divide and/or differentiate
Cells carry out normal functions
Why is it good if the cells are allowed to migrate out of an explant?
Retain morphological characteristics
What are the methods of mechanical dissociation?
Mincing,
Sieving
Pipetting
What enzymes are used to isolate primary tissue cells?
Trypsin Collagenase Hyaluronidase Protease DNAase
How do you separate blood cells?
Density centrifugation
Give some examples of primary non-haematopoietic cells
Liver, Endothelial cells Muscle Skin Nerves Fibroblasts Prostate
Give some examples of primary haematopoietic cells
Stem, progenitor cells T and B cells Monocyte Osteoblasts Dendritic cells Neutrophils Erythrocytes Megakaryocytes Platelets
What are the characteristics of cell lines?
Immortalised cells
Less limited (or unlimited) number of cell divisions
Phenotypically stable, defined population
Limitless ability
Easy to grow
Good reproducibility
Good model for basic science
What are the methods of production of cell lines?
Isolated from cancerous tissues
Immortalisation of healthy primary cultures
Genetic manipulation
How can you genetically manipulate cell lines?
Elongate telomeres
How do you elongate telomeres?
Introducing telomerase and inhibiting tumour suppressor proteins
What is the method for telomeric elongation?
SV40s T-antigen interacts with p53 and pRb
E-6 targets p53 for degredation and E7 binds to pRb, inactivating it
What do some cells need for immortalisation?
Introduction of the telomerase gene and inactivation of the pRb/p53
How are only the colonies with resistance able to survive?
Selection pressure is applied
What are 3D cell cultures?
Artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions
What are the disadvantages of 2D cell cultures?
Forced apical-basal polarity High stiffness Limited communication with other cells No diffusion of gradients Results not relevant to human physiology
What are the advantages of 2D cell cultures?
Simple, well established
Affordable
What are the advantages of 3D cell culture?
Adhesion in all three dimensions No forced polarity Variable stiffness Diffusion gradients of nutrients and waste products More relevant to human physiology
What are the disadvantages of 3D cell cultures?
More complex
More expensive
What are the two types of 3D cultures?
Spheroids and organoids
What are spheroids generated from?
Cell lines
What may spheroids exhibit?
Enhanced physiological responses
What do spheroids not do?
Undergo differentiation or self-organisation
What are organoids derived from?
PSCs, neonatal stem cells or adult stem cells
What do organoid cells spontaneously do?
Self organise into properly attenuated functional cell types and progenitors
What do organoids recap?
At least some function of the organ
What do patient derived organoids allow?
The study of cancer drug resistance
What is cell transfection?
Process by which foreign DNA is deliberately introduced into a eukaryotic cell through non-viral methods including both chemical and physical methods in the lab
What are the methods of cell transfection?
Lipofection
Electroporation
Nucleofection
Viral infection/transfection
What are the steps of lipofection?
Interaction with the cell membrane Taken up by endocytosis Release from the endosome Transport to the nucleus Entry to the nucleus is inefficient and may need mitosis
Why does lipofection work?
Liposomes have a net positive charge and plasma membranes are negatively charged
Why are liposomes potential drug carriers for drug delivery?
Can carry hydrophobic or hydrophilic drugs by attaching tissue specific antigens to the surface of the liposome which allows for targeted drug delivery
How does electroporation work?
Electric field applied to cells which increases their permeability - opens pores in the plasma membrane
What is nucleofection a combination of?
Electroporation and lipofection
What does viral infection/transduction exploit?
Mechanism of viral infection
What viruses are used for viral infection/transduction?
Retrovirus
Adenovirus
Most commonly lentivirus
What do viral transduction target cells need to do?
Express the viral receptor
What are the steps of viral transduction?
Create viral plasmid Carry out non-viral transfection to insert viral DNA into cell line Collect first supernatant Refrigerate Collect second supernatant Collect viral pellet after centrifuging Use for transduction or save in freezer
