Cells harvested from human blood and human tissues are being used to research basic biology, mechanisms of disease and to develop and test therapeutics. Harvesting cells from an organism, starts with a biopsy which depending on the tissue type can be performed by needle aspiration, scalpel, punch biopsy or laser. In the case of cell therapy, the harvested cells are the treatment but they may first be cultured and then harvested from cell culture before being administered to patient.
Cell harvesting is a step in the production of therapeutics like insulin that are grown in bacteria or antibodies produced in mammalian cell culture . In addition to the biomedical field, cell harvesting is a key step in bioprocessing of products like meat and leather made through cellular agriculture . To harvest cells from culture, cells, if they are adherent, are detached from the tissue culture surface and then they are separated from the culture medium by centrifugation or filters.
Harvesting from cell culture
Harvesting cells from culture is routinely done in research labs to refresh culture medium or split cell cultures into more culture dishes or flasks. Also, cells are harvested before analysis or extraction of cellular materials.
For cells that are grown in suspension, centrifugation can be the first step in cell harvesting. There needs to be enough centrifugal force to cause the dense solid cells to collect at the bottom of the centrifugation tube, but not so much as to destroy cell membranes . Disk stack centrifuges, originally developed for the dairy industry to separate fat from milk without shearing the fat particles, have been applied to cell culture cell harvesting because it is gentle on mammalian cells, which are particularly shear sensitive . Based on these, Culturefuge by Alpha Laval became the first fully hermetic cell culture centrifuge designed specifically for mammalian cell culture . In this system, cell culture fluid enters the centrifuge through a hollow inlet where is accelerates gradually as it moves upward to the disk stack, which is the rotating centrifuge bowl, where cells are separated from liquid culture medium . The liquid goes out an outlet on top of the bowl while the solids are ejected intermittently at the periphery of the bowl . These types of systems allow continuous separation of cells without contact with the external environment .
Depth filters are made of a porous filtration medium that traps particles or cells throughout the medium rather than particles remaining on the surface only. Depth filters can replace centrifugation as a method of capturing cells or can be used as a second step after centrifugation . Depth filters can be small single use devices that are connected to a pump system or they can be industrial-scale depth filtration systems . The filters are flushed with buffers to remove loose particles and flushed to recover the bound cells . For industrial size depth filters, flat sheets of are layered and stacked into cartridges inside stainless steel housings .
Before centrifugation or filtration, cells that adhere to surfaces must first be detached. Manual cell detachment by scraping is quick but may compromise cell viability and may not be feasible on an industrial scale . Trypsin, a protease enzyme commonly used in the lab for this purpose, works by cleaving the amino acids lysine and arginine in proteins that interact with the support surface . Trypsin is purified from pig pancreas but there are animal origin-free cell dissociation agents . Trysinization is often combined with shaking, combining mechanical and chemical methods . In microfluidic systems where cells are cultured on internal walls of channels, a strong fluid flow passing through results in detachment, but this method causes significant damage to cells . Non-enzymatic treatments like EDTA or citric saline are gentle detachment treatments . Thermoresponsive cell culture supports are coated in the polymer poly(N-isopropylacrylamide) (pNIPAAm) and cells detach upon temperature change . Similarly, pH-responsive polymers like chitosan, electro-responsive substrates and photo-responsive substrates can be used to release cells off supports .
Harvesting cells from blood and tissues
Hematopoietic stem cells and progenitor cells (HSPCs) are used for patients with certain blood or immune system disorders or patients undergoing cancer treatments . HSPCs are harvested from bone marrow using a needle with multiple side holes . HSPCs are harvested by puncturing the umbilical cord vein . HSPCs are not normally present in peripheral blood but are present if donors are pre-treated with granulocyte colony-stimulating factor before donating blood . Mesenchymal stem cells, also known as mesenchymal stromal cells (MSCs), can be harvested from umbilical cord and bone marrow . Lymphocytes and monocytes are separated out from blood samples for cell culture using density gradient centrifugation .
Cells from tissue biopsies are harvested after a process of agitation, enzymatic digestion and/or mechanical tissue dissociation and straining . Cells of interest can be isolated by detecting surface antibodies using fluorescence-activated cell sorting or magnetic-activated cell sorting, by differential ability to adhere to certain surfaces or by size using centrifugation or filtration . Laser capture microdissection allows the isolation of single cells . Using this technique, it is possible to collect several thousand cells . Microfluidics cell isolation, also known as lab-on-a-chip, uses electric, magnetic, gravity or mechanical force to sort cells as they pass through channels, chambers and valves . A single cell printer can take a mixed cell suspension and uses an optical system to detect single cells and deposit the live cells on surfaces for culture .
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