Mobilized peripheral blood is increasingly used as the source of hematopoietic stem cells for allogeneic transplantation, currently the only curative approach for sickle cell anemia. However, the safety and feasibility of stem cell mobilization in individuals with sickle cell trait (SCT) has not been documented. This study is a prospective controlled trial to evaluate the safety and feasibility of peripheral blood stem cell (PBSC) mobilization in 8 SCT subjects and 8 control subjects matched for age and race. Mobilization with filgrastim 10 g/kg subcutaneous daily for 5 days was followed by 12-L apheresis on the fifth day. Filgrastim administration was accompanied by similar symptoms in all subjects; no untoward adverse events occurred in either group, including sickle cell crises. CD34 ؉ cell mobilization response was not significantly different between SCT and control subjects. Median CD34 ؉ cell content was also similar in PBSCs collected from SCT versus control subjects, 6.8 versus 3.9 ؋10 6 CD34 ؉ cells/70 kg, P ‫؍‬ .165. Red cell depletion from SCT products was not possible by using hydroxyethyl starch sedimentation but was achievable with ammonium chloride lysis. There was no evidence of gelling of SCT products after thaw, and no difference in cell recovery was seen among red cell-depleted versus nondepleted products. Cryopreservation in 5% dimethyl sulfoxide/6% pentastarch was associated with superior cell recovery (both SCT and control subjects) compared with 10% dimethyl sulfoxide (P ‫؍‬ .001). The study concluded that filgrastim mobilization, large volume apheresis, processing, and cryopreservation appears to be safe in donors with SCT, allowing PBSC use for transplantation in patients with sickle cell anemia. (Blood. 2002;99:850-855)

BackgroundBone marrow stromal cells (BMSCs) are being used to treat a variety of conditions. For many applications a supply of cryopreserved products that can be used for acute therapy is needed. The establishment of a bank of BMSC products from healthy third party donors is described.MethodsThe recruitment of healthy subjects willing to donate marrow for BMSC production and the Good Manufacturing Practices (GMP) used for assessing potential donors, collecting marrow, culturing BMSCs and BMSC cryopreservation are described.ResultsSeventeen subjects were enrolled in our marrow collection protocol for BMSC production. Six of the 17 subjects were found to be ineligible during the donor screening process and one became ill and their donation was cancelled. Approximately 12 ml of marrow was aspirated from one posterior iliac crest of 10 donors; one donor donated twice. The BMSCs were initially cultured in T-75 flasks and then expanded for three passages in multilayer cell factories. The final BMSC product was packaged into units of 100 × 106 viable cells, cryopreserved and stored in a vapor phase liquid nitrogen tank under continuous monitoring. BMSC products meeting all lot release criteria were obtained from 8 of the 11 marrow collections. The rate of growth of the primary cultures was similar for all products except those generated from the two oldest donors. One lot did not meet the criteria for final release; its CD34 antigen expression was greater than the cut off set at 5%. The mean number of BMSC units obtained from each donor was 17 and ranged from 3 to 40.ConclusionsThe production of large numbers of BMSCs from bone marrow aspirates of healthy donors is feasible, but is limited by the high number of donors that did not meet eligibility criteria and products that did not meet lot release criteria.

Background Cell selection is an important part of manufacturing cellular therapies. A new highly automated instrument, the CliniMACS Prodigy, was evaluated for the selection of CD34+ cells from mobilized peripheral blood stem cell (PBSC) concentrates using monoclonal antibodies conjugated to para-magnetic particles. Methods PBSCs were collected by apheresis from 36 healthy subjects given G-CSF or G-CSF plus plerixafor. CD34+ cells from 11 PBSC concentrates were isolated with the automated CliniMACS Prodigy and 25 with the semi-automated CliniMACS Plus Instrument. Results The proportion of CD34+ cells in the selected products obtained with the two instruments was similar; 93.6±2.6% for the automated and 95.7±3.3% for the semi-automated instrument (p > 0.05). The recovery of CD34+ cells from PBSC concentrates was less for the automated than the semi-automated instrument (51.4±8.2% versus 65.1±15.7%; p=0.019). The selected products from both instruments contained few and similar quantities of platelets and red blood cells. The depletion of CD3+ cells was less with the automated instrument (4.34±0.2 log depletion versus 5.20±0.35 log depletion; p < 1 × 10−6). Removal of platelets from PBSC concentrates by washing was associated with better CD34+ cell recovery. We explored the reasons for lower CD34+ cell recovery by the Prodigy and found that the non-selected cells for the Prodigy contained more platelets than those for the CliniMACS Plus. Conclusions CD34+ cells can be effectively selected from mobilized PBSC concentrates with the CliniMAC Prodigy, but the recovery of CD34+ cells and depletion of CD3+ cells was lower than with the semi-automated CliniMACS Plus Instrument.

BackgroundOvarian cancer has no definitive second line therapeutic options, and largely recurs in the peritoneal cavity. Locoregional immune therapy using both interferons and monocytes can be used as a novel approach. Interferons have both cytostatic and cytotoxic properties, while monocytes stimulated with interferons have potent cytotoxic properties. Due to the highly immune suppressive properties of ovarian cancer, ex vivo stimulation of autologous patient monocytes with interferons and infusion of all three agents intraperitoneally (IP) can provide a strong pro-inflammatory environment at the site of disease to kill malignant cells.MethodsPatient monocytes are isolated through counterflow elutriation and stimulated ex vivo with interferons and infused IP through a semi-permanent catheter. We have designed a standard 3 + 3 dose escalation study to explore the highest tolerated dose of interferons and monocytes infused IP in patients with chemotherapy resistant ovarian cancer. Secondary outcome measurements of changes in the peripheral blood immune compartment and plasma cytokines will be studied for correlations of response.DiscussionWe have developed a novel immunotherapy focused on the innate immune system for the treatment of ovarian cancer. We have combined the use of autologous monocytes and interferons alpha and gamma for local–regional administration directly into the peritoneal cavity. This therapy is highly unique in that it is the first study of its type using only components of the innate immune system for the locoregional delivery consisting of autologous monocytes and dual interferons alpha and gamma.Trial Registration ClinicalTrials.gov Identifier: NCT02948426, registered on October 28, 2016. https://clinicaltrials.gov/ct2/show/NCT02948426