Combined treatment with doxorubicin-loaded NBs and ESWs is a promising drug delivery tool for ATC treatment with the possibility of using lower doxorubicin doses and thus limiting its systemic side effects.

BackgroundCytokine-induced killer (CIK) cells are a very promising cell population raising growing interest in the field of cellular antitumor therapy. The aim of our study was to validate the most advantageous expansion method for this advanced therapy medicinal product (ATMP) and to translate it from preclinical field to good manufacturing practices (GMP). GMP ensures that ATMP are consistently produced and controlled to the quality standards required to their intended use. For this reason, the use of the xenogenic sera tended to be minimized by GMP for their high variability and the associated risk of transmitting infectious agents.ResultsWe decided to replace Fetal Bovine Serum (FBS), largely used as medium supplement for CIKs expansion, with other culture media. Firstly, Human Serum (HS) and Human Pool Plasma (HPP) were tested as medium supplements giving not compliant results to acceptance criteria, established for CIKs, probably for the great batch to batch variability. Consequently, we decided to test three different serum free expansion media: X-VIVO 15, (largely used by other groups) and Tex Macs and Cell Genix GMP SCGM: two GMP manufactured media. We performed a validation consisting in three run-sand even if the small number of experiments didn’t permit us to obtained statistical results we demonstrated that both X-VIVO 15 and Tex Macs fulfilled the quality standards in terms of cellular growth, viability and identity while Cell Genix GMP SCGM resulted not compliant as it caused some technical problems such as high mortality.ConclusionIn conclusion, these preclinical validation data lay the bases for a GMP-compliant process to improve the CIKs expansion method.

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.

Mesenchymal stem cells (MSCs) are classified as advanced therapy medicinal products, a new category of GMP (good manufacturing practice)-compliant medicines for clinical use. We isolated MSCs from 5 bone marrow (BM) samples using human platelet lysate (HPL) instead of foetal bovine serum (FBS). We used a new method of HPL production consisting of treating platelet (PLTs) pools with Ca-Gluconate to form a gel clot, then mechanically squeezing to release growth factors. We compared the new HPL (HPL-S) with the standard (HPL-E) obtained by freezing/thawing cycles and by adding heparin. HPL-S had not PLTs and fibrinogen but the quantity of proteins and growth factors was comparable to HPL-E. Therefore, HPL-S needed fewer production steps to be in compliance with GMP conditions. The number of colonies forming unit-fibroblasts (CFU-F) and the maintenance of stem markers showed no significant differences between MSCs with HPL-E and HPL-S. The cumulative population doubling was higher in MSCs with HPL-E in the earlier passages, but we observed an inverted trend of cell growth at the fourth passage. Immunophenotypic analysis showed a significant lower expression of HLA-DR in the MSCs with HPL-S (1.30%) than HPL-E (14.10%). In conclusion, we demonstrated that HPL-S is an effective alternative for MSC production under GMP conditions.

Objective: 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 pol 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. Methods: 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 pol gene, NANOG, OCT-4, SOX-2, and GAPDH expression was quantified by real-time PCR in MSCs during the expansion. Results: 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. Conclusions: Our findings showed that HERV-K and H were concurrently expressed with pluripotency biomarkers NANOG, OCT-4, and SOX-2.