Tissue culture plates and flasks

As the first step in scale-up is (or should be) to produce enough cells to lay down a cell bank in liquid nitrogen PniUKol 2 describes the procedure for growing cells in stationary monolayer cultures with the purpose of creating a cell bank. 

Growth of hybridoma cells in stationary monolayer cultures 

Add 1 ml DMEM (or similar medium) to each well of a multidish (4 x 1 ml wells with a 1,9 cm surface area) and add minimum of 2 (preferably 5) xio cells. Cover and place in a 37 C CO incubator for four days. 

Harvest cells and medium from wells, giving a slight tap to dislodge cells and get them in homogeneous suspension. Pool cells, cany out a haemocytometer count, and add to a 25 cm flask containing 7 ml pre-warmed medium at 2-5 x 10 cells/ml. Gas with 5% C02 and incubate for a further four days. 

Harvest the culture expected yield 5-7 X 10 cells. Add to a 75/80 cm flask 30 ml medium and 1-2 xio cells. There should be enough cells to set up at least three flasks. Gas with 5% CO and incubate for a further four days. 

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Althou hybridoma cells are spherical and well adapted to suspension culture they do grow well in stationary culture resting on the substrate, sometimes with very light attachment. This means that a wide selection of culture vessels and systems are available for their culture ranging from a simple tissue culture plate or flask to highly sophisticated bioreactors with full instrumentation to control the physiological environment (1-3). Commercially hybridoma cell lines are grown at scales up to 2000 litres and beyond in culture units scaled-up from laboratory size vessels. Laboratory, pilot, and production scale is ill-defined so in this chapter laboratoiy scale will be taken as 10 litre volume cultures, and below. 

Fig. 3.2(a). Vessels in which cells may be cultured. At the top left are three sizes of flask (125, 75 and 25 cm2) and at the bottom left three sizes of dish (9.5 and 3 cm diameter). To the right are shown 6, 12 and 24-well tissue culture trays and a 96-well microtitre plate. At the bottom right is an 8-chamber culture slide, (b) Millicell inserts for 6-well TC trays provide a semi-permeable growth surface. 

Note the monocytes will differentiate into adherent macrophages by day 7 of incubation. To harvest cells, discard the culture medium and wash once with 25 mL of cold PBS without Ca++ or Mg++. Add 20 mL of fresh cold PBS to the flask and incubate on ice until cells begin to round-up and detach (usually 15-20 min) (see Note 14). Gently scrape cells into the PBS with a cell scraper and transfer the cell suspension to a 50-mL conical centrifuge tube. Pellet the cells by centrifugation at 500 x g for 5 min. Resuspend cells in 5-10 mL of RPMI plus 10% FBS and 50 ng/mL M-CSF. Count cells, add medium to adjust to the desired cell concentration, and re-plate in a tissue culture plate (see Note 15). Incubate at 37 °C in 5% CO2 for at least 6 h to allow macrophages to adhere. 

Tissue culture-treated plastic flasks, dishes, and 24-well plates. 

Tissue culture flasks (T-75, Cat. No. 10-126-41), six-well tissue culture plates (Cat. No. 08-772-lB), sterile polypropylene conical tubes (50-ml capacity, Cat. No. 05-538-55A), sterile polypropylene round-bottom tubes (5-ml capacity. Cat. No. 14-959-lOA), and sterile conical-bottom microfuge tubes (1.5-ml capacity. Cat. No. 05-664-63) are obtained from Fisher Scientific. Disposable 1-ml syringes (Cat. No. 309602) and 30-gauge hypodermic needles (Cat. No. 5106) are purchased from Becton-Dickinson. A Hamilton microliter syringe (50-//1 capacity. Cat. No. 80501) is obtained from Hamilton Company. Micropipette tips (MC-50) are purchased from West Coast Scientific. The hand-held pipettor (Cat. No. P20) is obtained from Gilson Medical Electronics. 

Three different approaches for the cultivation of isolated hematopoietic cells have been described, the static, the stirred and the immobilized culture. Static cultivation takes place in very simple culture systems like well plates, tissue-culture flasks or gas-permeable culture bags [62, 63]. As the first two systems do not allow cell cultivation on a clinical scale, the latter is actually the most often used technique for stem cell expansion. All these systems have the advantage of being easy to handle, single-use devices, which enable an uncomplicated cell harvest. But all of them do not offer possibilities for process control or continuous feeding. This causes variations in culture conditions during fermentation (e.g., oxygen tension, pH, substrate, metabolite and cytokine concentrations). 

Cells to be tested are grown in a tissue culture dish (not a flask, which would be difficult to scrape) until confluent. Most of the growth medium is removed, and an artificial linear wound is made in the monolayer, by using, e.g. a sterile pipet tip, or a rubber policeman . Care has to be taken to clean all the wounded area from cells, because in case an uneven scraper is used, which leaves behind some attached cells, their presence at the end of the experiment may complicate the interpretation of the results. The plate is then rinsed several times with PBS to remove all the debris, and the test medium (containing dilutions of the supposed migration-... 

Because most routine cell types were originally grown on glass, the first commercially available tissue culture surface was modelled after glass chemistry. Conventional tissue culture surfaces therefore are hydrophilic and have an oxygenated chemistry and a net-negative surface charge. This chemistry is basically the same whether the treatment process is produced by corona or plasma. This is the routine surface that is commercially available from a number of different suppliers on plastic dishes, flasks, plates and roller bottles. 

Chamber slides are best for cells that require tissue culture grade plastic as a substrate. Lab-Tek chamber slides come with such a slide and they come in a range of well sizes, but a very practical size is the 4-well plate (Fig. 4.7). The microscope slide base is made of Permanox, the same plastic used for plastic petri dish bottoms so that the cells grow like they do on petri dishes or flasks. The nice thing about the chamber slides is that wells on the slide do not need be removed until after immunocytochemistry processing. The wells can be removed just before mounting a... 

Human gingival fibroblasts (HGF) were maintained according to the method of Elvin and Evans [19]. Stock cultures were recovered from liquid nitrogen and plated at 200,000 cells per 25 cm tissue-culture flask in a-DMEM with 10% foetal bovine serum and 1% antibiotics. 

The culture of stem cells is traditionally and usually performed on fiat two-dimensional surfaces such as tissue culture flasks (T-flasks), well plates, or gas-permeable blood bags consisting of a single unstirred compartment where nutrients diffuse to cells. Gas exchange (e.g., oxygen and carbon dioxide) occurs at the medium/gas interface. These systems are widely used for research purposes because of their simplicity, ease of handling, and relatively low cost. 

Since the first in vitro reconstruction of the in vivo hematopoietic microenvironment to culture HSPCs by Dexter et al. (1973), which was later adapted for human cells (Gartner and Kaplan, 1980), hematopoietic cell cultures have been typically performed in multiwell plates, or tissue culture flasks made of polystyrene suitable for cell culture. Gas permeable culture bags are also currently used (Haylock et al., 1992 Lemoh et al., 1992). 

Fig. 3 Tissue engineering culture environment designs (A) plate (B) spinner flask (C) perfusion (D) hollow fiber and (E) rotating wall bioreactor.

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