Q&A follow-up: Why are we still using FBS in our processes?
Q&A follow up of the live webinar event.
Thank you to everyone who attended the webinar titled ‘Why are we still using FBS in our processes?’ on June 20, 2017.
Below is a follow-up of the Q&A session.
What are you doing about monitoring the stem cell-specific fraction during expansion?
Dr Qasim Rafiq: We perform the relevant quality and functional assays to ensure the cells we are expanding can be characterized as Human Mesenchymal Stem Cells (hMSCs).
During scale up for stem cell production processes, are expansion steps typically at fixed intervals?
Dr Qasim Rafiq: Production runs for stem cell bioprocesses will typically depend on the specific process requirements. In our studies, we run reactors for multiple days and harvest when we believe we have maximized productivity.
How does the dexamethasone effect on human platelet lysate (HPL)?
Dr Qasim Rafiq: This is not something we have investigated.
How was the total viable cell number calculated?
Dr Qasim Rafiq: Total viable cell number was calculated by taking daily samples and analyzing acridine orange uptake and DAPI exclusion.
What volume of HPL is currently available or would be required to support cell therapy manufacturing and to overcome the limited global GMP-grade fetal bovine serum (FBS) supply?
Dr Qasim Rafiq: This is a very interesting question and will ultimately depend on supply requirements. Availability of an ethically sourced supplement is likely to be achievable with HPL compared with FBS. With respect to the amount needed to support cell therapy requirements will depend on the optimal concentration required which is likely to be process specific.
What method is used to get cells off of the microcarrier beads for colony formation analyses?
Dr Qasim Rafiq: We use a 2-step harvesting process which involves detaching the cells from the microcarriers and then a filtration step to separate the microcarriers and the cells. Single cells are then plated for the CFU-f analysis.
Do you have any experiences or comments on the performance of animal component free or defined media in these applications and assays, compared to FBS and HPL?
Dr Qasim Rafiq: We have investigated the use of HPL and multiple commercially available serum/xeno-free media. This will be the focus of a future publication. In brief, we find that some xeno-free media facilitate increased levels of cell proliferation whilst retaining the critical quality attributes.
Have you tried HPL from other vendors and if yes, have you experienced different performances?
Dr Qasim Rafiq: We have not investigated HPL from other vendors, but it is something we will be pursuing moving forward.
On the metabolism slide for glucose lactate measurements, what happens at 72 hours?
Dr Qasim Rafiq: This is where a medium exchange was performed (sample taken before medium exchange and afterwards).
Did you try HPL for any other cells other than hMSCs?
Dr Qasim Rafiq: We have not investigated the use of HPL with other cell types, but this has been the focus of other peer-reviewed studies.
How easy and cost-effective is it to scale-up processes using HPL compared with FBS?
Dr Qasim Rafiq: We have found that the ability to scale a process in HPL is equivalent to that of FBS from a technical perspective. Costs need to be considered carefully, but we have found with improved consistency and yield, overall costs can be significantly reduced as the process is scaled.
Is the HPL similar to the platelet rich plasma?
Cook Regentec: HPL and PRP are similar, with both derived from platelets. PRP is comprised of concentrated, intact platelets, and unlike HPL is not commonly used as a culture supplement. Current variants of PRP may be leukocyte enriched or depleted.
Cook Regentec: Pharma organizations are really good at evaluating a potential manufacturing reagent for both safety and function. The material that works the best with an acceptable risk profile will end up in the manufacturing process. There are certainly risk and regulatory elements that need to be considered when using a human derived material. Regulators have approved both clinical studies using human derived manufacturing reagents as well as drug products derived from human materials. It’s good to discuss these questions with the responsible regulator.
IS HPL virally inactivated?
Viral risk is managed through donor testing and screening as defined for therapeutic blood product donations.
What is the shelf life of HPL once thawed?
Cook Regentec: The shelf life once thawed for HPL varies by vendor. Consult with your HPL vendor for proper instructions for use.
Can HPL be used in cryopreservation in place of FBS in freezing media?
Cook Regentec: This is not something we have investigated but Cook Regentec notes that HPL has been evaluated as a cryopreservation media additive, with generally good results. Cook Regentec research has shown cells cryopreserved in media supplemented with HPL has better viability and recovery than those preserved in standard medium (Thirumala et al., 2015 ISCT).