Microbioreactor-Based Production of Anchorage-Dependent Mesenchymal Stromal Cells Primed for Acute Respiratory Distress Syndrome

We study how cells interact with their environment, including when producing or manufacturing cells as therapies. Here, we address questions about how anchorage-dependent cells like MSCs, mesenchymal stromal, or stem cells, can be produced to overcome some barriers to clinical translation. Growing recognition that cell population variability affects both the consistency and potency of the final cell product for every batch or person.

These challenges can even affect the regulatory approval for some cell therapies. We target a gap that can achieve a high number of cells per milliliter of fluid at high control of the environment and in a way that can be scaled up. This protocol provides a way to include inline environmental monitoring for cells that are anchorage-dependent like MSCs.

That process control is an advantage over flasks that are still used in most lab and clinical production. There are several open questions for MSCs and other cell types that are sensitive to mechanical cues. For example, how can this kind of approach help engineer cells to address other disease states?

To begin, remove the clear plastic cap connected to bottle LL in the bottle rack. Using a new sterile 50-milliliter syringe without a plunger, connect the nozzle to bottle LL.Pour the sterile filtered water into the syringe barrel and inject it into bottle LL.Similarly, fill bottle L with 25 milliliters of low-glucose DMEM without FBS, bottle R with 25 milliliters of sterile PBS, and bottle RR with eight milliliters of anti-adherence rinse solution. Repeat the procedure to prepare the required number of consumables for the experiment.

Next, perform a self-test on each pod to be used in the experiment. Once the pod has successfully passed all checks from the self-test, remove the self-test cassettes and place them into light-protected storage bags. Mount the consumable to the bioreactor.

Remove the green tape from the consumable valves, and install the consumable in the pod. Reinstall the clamps and check for any hissing sound to confirm a secure seal. Connect the white pressure sensor line, the red air pressure line, and the blue vacuum line between the consumable and the base station or pod.

Confirm that the side injection port is clamped shut using a white teardrop clamp before initiating a new experiment. Click the Flush Bottles icon in the software interface. Enable the radio button for bottle L, select Output to Waste, then click Start Flush MSCserum.

In the Bottle Contents window, indicate that MSCserum is located in bottle R, which contains PBS. Then navigate to Manual Ops and perform three Eject Wash cycles on the waste bottle. In the Bottle Contents window indicate that MSCserum is now located in bottle RR, which contains the anti-adherence rinse solution.

Go back to Manual Ops and perform one Eject Wash cycle on the waste bottle. After 30 minutes, rinse out the consumable with PBS. Empty the reactor to await cell and microcarrier injection.

In the Manual Ops panel click Reinoculate. Leave the system paused at the stage where the dashboard displays a prompt to inoculate the reactor. Next, pipette a calculated volume of resuspended cells into a 15-milliliter conical tube containing microcarriers for each condition.

Top up each tube to between 2.2 and 2.5 milliliters with fresh, warm human mesenchymal stem cell media. Working with one or two consumables at a time, remove them from the base station and bring them into the biosafety cabinet. Withdraw the cell and microcarrier mixture into a five-milliliter syringe using a green four-inch blunt needle.

Inject the mixture into the reactor. Grasp the injection port line near the base of the cassette using caution not to detach the line. Using a rolling motion between thumb and index finger, twist the injection port line for two full rotations to create a knotted kink in the line.

Tuck the knot into the cut-out space near the base of the injection port and secure it with a small piece of laboratory tape. Then return the consumable to the base station. After repeating the injection for all consumables, navigate through the software interface to indicate that inoculation is complete.

When the software returns to the homepage, remove the white clamp from the consumable output lines. Enter Manual Ops and perform the Eject Excess function with the Output Port set to P for perfusion. Enable Static Mixing mode using a cycle of 1, 680 seconds on and 120 seconds off, and set Mixing Frequency to five hertz.

To begin cell harvesting, use a one-milliliter syringe to draw up 200 microliters of Pronase and approximately 300 to 400 microliters of air. Unscrew the cap on the side port injection line and spray the needleless valve connector with 70%ethanol. Remove the tape from the knotted side port injection line, untie the knot, and unclamp the white teardrop clamp.

Now, connect the one-milliliter syringe and gently inject the Pronase into the reactor. Chase the liquid with air to ensure complete delivery. Reengage the teardrop clamp and recap the injection line.

Then return the consumable to the pod, reconnect all lines, remove the white C-clamp and resume mixing. Place the waste bottle and media source bottle on ice. Start a five-minute timer and allow the Pronase to digest the microcarriers in the reactor.

After five minutes, verify that the microcarriers have degraded by checking their absence in the reactor. Harvest the cells by entering Manual Ops, set the number of cycles to three, choose Output Port W for waste, and click on Eject Wash. After the Eject Wash cycles are complete, transfer the consumable to the biosafety cabinet.

Then withdraw the contents of the waste bottle using a 10 or 20-milliliter syringe. Dispense the contents of the waste bottle into a 15-milliliter conical centrifuge tube. Centrifuge the cells at 300g for five minutes.

Aspirate the supernatant and evaluate the cell pellet size. Environmental conditions, including pH, temperature, dissolved oxygen, and carbon dioxide, were stably maintained throughout the 10-day culture period in the microbioreactor system. The pH variation was significantly lower in the microbioreactor compared to the tissue culture polystyrene flask condition.

Cell yield was higher in the microbioreactor than in the T25 flask while maintaining similar cell viability. Of the 14 measured potency-associated critical quality attributes, nine showed significantly higher mRNA expression in the microbioreactor condition relative to the T75 flask.