Cytokine-Induced Killer (CIK) Cells, In Vitro Expanded under Good Manufacturing Process (GMP) Conditions, Remain Stable over Time after Cryopreservation

Cytokine-induced killer (CIK) cells are advanced therapy medicinal products, so their production and freezing process has to be validated before their clinical use, to verify their stability as a drug formulation according to the good manufacturing practice (GMP) guidelines. We designed a stability program for our GMP-manufactured CIK cells, evaluating the viability, identity and potency of cryopreserved CIK cells at varying time periods from freezing, and compared them with fresh CIK cells. We evaluated the effects of the cryopreservation method, transportation, and the length of time of different process phases (pre-freezing, freezing and post-thawing) on the stability of CIK cells. This included a worst case for each stage. The expanded CIK cells were viable for up to 30 min from the addition of the freezing solution, when transported on dry ice within 48 h once frozen, within 60 min from thawing and from 12 months of freezing while preserving their cytotoxic effects. The reference samples, cryopreserved simultaneously in tubes and following the same method, were considered representative of the batch and useful in the case of further analysis. Data obtained from this drug stability program can inform the accurate use of CIK cells in clinical settings

Evaluating Ecological Impact and Sustainability in the Manufacturing of Advanced Therapies: Comparative Analysis of Greenhouse Gas Emissions in the Production of ATMPs in Open and Closed Systems

The primary aim of this systematic analysis is to highlight opportunities to improve the environmental impact of advanced therapy medicinal products (ATMP) manufacturing. We have compared the Greenhouse Gas (GHG) emissions expressed in CO2eq of a classic clean room open system (AinB) Cell Factory versus a comparable closed system equipped with isolators (AinD). We have therefore outlined a theoretical situation to simulate the use of a closed system with an equivalent production output to that obtained in the Cell Factory (CF) of the Regina Margherita Children’s Hospital. Open and closed systems for ATMPs have been compared as regards energy requirements, ecological footprints, and costs by analyzing a hypothetic cell production cycle of 21 days. The results demonstrate energy saving and a reduction of 52% in GHG emissions using closed systems per process cycle. Moreover, a reduction in production costs in an isolator setting is also evident. This study shows that the closed system solution has evident advantages compared with the open one

Human Endogenous Retrovirus-H and K Expression in Human Mesenchymal Stem Cells as Potential Markers of Stemness

&lt;b&gt;&lt;i&gt;Objective:&lt;/i&gt;&lt;/b&gt; The human endogenous retroviruses (HERVs) are endogenous retroviruses that were inserted into the germ cell DNA of humans over 30 million years ago. Insertion of HERVs into the chromosomal DNA can influence a number of host genes in various modes during human evolution and their proviral long terminal repeats can participate in the transcriptional regulation of various cellular genes. Our aim was to evaluate the &lt;i&gt;pol&lt;/i&gt; gene expression of HERV-K and HERV-H in mesenchymal stem cells (MSCs) in relation with the expression of stemness genes such as NANOG, OCT-4, and SOX-2. &lt;b&gt;&lt;i&gt;Methods:&lt;/i&gt;&lt;/b&gt; MSCs were isolated from bone marrow of healthy donors and expanded until the 5th passage in α-MEM with 10% fetal bovine serum. HERV-K, HERV-H &lt;i&gt;pol&lt;/i&gt; gene, NANOG, OCT-4, SOX-2, and GAPDH expression was quantified by real-time PCR in MSCs during the expansion. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; HERV-K and HERV-H expression was always higher at p1 compared to other passages and this difference reached a high statistical significance when passage p1 was compared with passage 3. In addition, NANOG, OCT-4, and SOX-2 expression at p1 was significantly higher than their expression at p3. Pearson’s test demonstrated a strong correlation between the expression of HERV-K and HERV-H and the expression of NANOG, OCT-4, and SOX-2. &lt;b&gt;&lt;i&gt;Conclusions:&lt;/i&gt;&lt;/b&gt; Our findings showed that HERV-K and H were concurrently expressed with pluripotency biomarkers NANOG, OCT-4, and SOX-2.</jats:p