3.2.5.1. Determination of cell viability by analysis of metabolic activity (AlamarBlue assay)
To analyse the viability of cultured and encapsulated cells an AlamarBlue assay was performed which displays the metabolic activity of analysed cells. In proliferating cells specifically the ratios of NADPH/NADP, NADH/NAD, FADH2/FAD and FMNH2/FMN are increasing. This metabolic activity is measured in the AlamarBlue assay.
The substrate AlamarBlue is an oxidation-reduction (REDOX) indicator that undergoes a colorimetric change and yields a fluorescent signal in response to metabolic activity. Reduction causes a colour change of the oxidized form, resazurin (non-fluorescent, blue) to the reduced form, resorufin (fluorescent, red). AlamarBlue is taken up by the cell and is reduced by the metabolic intermediates; thus can be used to monitor cell proliferation by a measurable shift in colour. There are two ways to monitor AlamarBluereduction: by measuring absorbance in a spectrophotometer or by measuring fluorescence.
Method:
Approximately 200 capsules were transferred into a 6-well plate. After medium exchange and capsule sedimentation, the supernatant was removed and cells were washed by adding fresh medium. The washing procedure was repeated a second time. In order not to damage encapsulated cells, 200 µl filter tips with wide openings were used when handling capsule suspensions. 10 capsules were pipetted in triplicate into a black 96-well plate. Cell culture medium was pipetted as sample blank in triplicate into the 96-well plate. AlamarBlueTM reagent was pipetted into all wells containing samples or blanks. 1.5 mM resurofin stock solution (which served as a
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fluorescent standard, stored at -20°C) was diluted to 37.5 µM, 12.5 µM, 4.17 µM 1.39 µM, 0.463 µM and 0.154 µM. Each standard dilution was pipetted in triplicates onto the 96-well plate. The 96-well plate was carefully agitated and incubated at 37°C, 5% CO2 saturation and 95% relative humidity for 4 h. In viable (encapsulated) cells resazurin is converted to resorufin. The amount of formed resorufin was analysed using the Tecan GeniosTM device. The resorufin standard enables calculation of the amount of resorufin, which is generated by cells using AlamarBlueTM as a substrate.
3.2.5.2. Determination of cell viability by analysing cell membrane integrity (TrypanBlue assay)
In order to analyse viability of cultured cells and of cells which should be encapsulated, a TrypanBlue assay was performed. In the TrypanBlue assay dead cells are stained blue and this can be analysed by using a light microscope. The dye can only enter cells via damaged or disordered membranes. Thus, based on the integrity of cell membranes, the TrypanBlue assay allows to distinguish between live and dead cells.
Method:
Cells were trypsinised. An aliquot of the cell suspension was centrifuged for 3 min at 260 x g. The cells were resuspended in PBS. The cell suspension was mixed carefully to avoid inhomogeneity. A sample of the cell suspension was transferred into an Eppendorf tube containing TrypanBlue reagent. Then the 1:1 mixture was incubated for 2 min at RT. An aliquot of the mixture was transferred into a Neubauer cell counting chamber. Four square areas with cells were analysed by counting the cell number of each large square separately. Additionally, the blue stained cells of each square were counted. The total cell number and the number of living cells was finally calculated.
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3.2.5.3. Determination of cell viability by analysing intracellular esterase activity and membrane integrity by co-staining with calcein and propidium iodide
In order to analyse cell viability, encapsulated cells were co-stained with calcein and propidium iodide. Cells were then released from capsules and subsequently analysed by FACS. This method is utilised for simultaneous fluorescence staining of live and dead cells. Calcein-AM, which is a acetomethylester of calcein, is highly lipophilic and permeates intact cell membranes. While calcein-AM itself is not a fluorescent molecule, the calcein generated from calcein-AM by intracellular esterases emits strong green fluorescence (exitation: 490 nm, emission: 515 nm), which is the indicator for viable cells. In contrast, propidium iodide (PI) only stains dead cells. PI, a nuclei staining dye, can`t pass intact membranes of viable cells. It reaches the nucleus by passing through disordered areas of the membrane of dead cells and finally, intercalates within the DNA double helix. PI emits red fluorescence (excitation: 535 nm, emission: 617 nm).
Method:
Capsules to be analysed were pipetted into a 6-well plate. Medium was removed and capsules were washed with SFMI medium twice. This medium is a serum-free medium for culturing HEK293 cells. This medium resolves SCS capsules. For lysis, capsules were incubated in SFMI medium on a horizontally shaker (~20-30 rpm) for 90 to 180 min in a cell culture incubator at 37°C. After 90 minutes, the progression of lysis was microscopically controlled and capsules were then sheared mechanically by pipetting up and down ten times with a 1000 µl pipette. After incubation, the capsules were sheared again. Depending on the density within the capsules, further separation of cell aggregates was required. Resolved capsules were transferred to an Eppendorf tube and centrifuged for 5 min with 2000 rpm. Supernatant was removed carefully to avoid destruction of the instable pellets. Pellets were resuspended in Biotase (0,0042 % protease / 0,02 % EDTA (w/v), Biochrome) or collagenase (0.5 mg/ml type 3, Worthington) and incubated for 10 min at 37°C on a thermoshaker (500 rpm). Subsequently, the process was stopped by adding two volumes of NM. Cells were centrifuged with 2000 rpm and washed twice with PBS.
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For live/dead staining the single cell suspension stained with calcein and propidium iodide at a final concentration of 0.5 µM and 0.75 µM respectively. Cells were finally analysed by FACS using a FACScalibur (BD Biosciences).