OPTIMIZATION OF CONCENTRATING UC-MSC CONDITIONED MEDIUM USING CENTRIFUGAL FILTRATION

• Published in: Cytotherapy (Oxford, England) England), 2024-06, Vol.26 (6), p.S62-S62
• Main Authors: Chouw, A., Sartika, C.R., Facicilia, G., Shyallala, R.A., Arofah, A.N., Agustina, R., Pratiwi, A.R.
• Format: Article
• Language: English
• Subjects: Concentrated; Conditioned Medium; Mesenchymal Stem Cells
• ISSN: 1465-3249; 1477-2566
• DOI: 10.1016/j.jcyt.2024.03.110

The devastating long-term outcomes of Traumatic Brain Injury (TBI) underscore the critical need for innovative interventions that go beyond symptom management and offer genuine neuro-restoration capabilities. TBI is intricately linked to the dynamic interplay of immunomodulatory cytokines, notably IL-4 and IL-10, orchestrating a complex regulatory network. Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSCs) derived conditioned medium (CM) has diverse pro- and anti-inflammatory factors, which have the potential in helping nerve regeneration in TBI. While UC-MSC CM therapeutic potential for neurological disorders are undeniable, its clinical translation faces the formidable hurdle to penetrate the Blood-Brain Barrier. One promising approach involves concentrating the bioactive components of CM, thereby enabling efficient delivery to the targeted neuronal milieu. Here, we aim to concentrate the UC-MSC CM using 2 types of filter sizes. UC-MSCs were cultured at passage 5 until reached >80% confluency. Then, UC-MSC CM was harvested and further processed with a Centrifugal Concentrator using the 3 kDa and 10 kDa cut-off filters. As much as 20 mL of UC-MSC CM were transferred to each column and then centrifuged at 2400xg for 40 minutes at 4°C. Both the retentate and the filtrate were sampled for sterility, endotoxin, mycoplasma, cytokine (IL-4, IL-10, IL-6, TNF-α), and total protein analysis. Results showed that although the total protein analysis of each group was not of significant difference (167-188 mg/mL, p>0.05), the concentration of both pro- and anti-inflammatory cytokines recovery is highly concentrated using 3-kDa filter size compared to 10-kDa. Using 3-kDa filter size, approximately 83.1% and 74.9% recovery of IL-10 and IL-4 can be achieved, respectively. Additionally, the concentration of IL-6 and TNF-α can be preserved up to 97.7% and 80.9%, respectively. All samples were negative for microbial contamination, indicating an effective sterilization procedure. The result suggested that to fraction only the anti-inflammatory cytokines, differential centrifugation should be applied by spinning down the UC-MSC CM using filter columns with greater cut off size than 10 kDa and then spinning it down using the 3 kDa cut off filter. In conclusion, our study reveals that the centrifugal filtration effectively concentrated key anti-inflammatory cytokines in UC-MSCs-derived CM, potentially enhancing neuroprotective potential for TBI therapy.