Fig. 30.1
The isolation method of placental MSC
When the Brisbane cGMP facility became available (QGen) procedures were performed in clean rooms at Q-Gen, a Therapeutic Goods Administration (TGA) licensed facility (Licence #174586). All cell products were tested according to the TGA’s product release criteria which include maternal placental donor exclusion criteria and release criteria at cryopreservation including release from manufacturing (see below). Maternal placental donor guidelines were those of the Australian Red Cross Blood Services (ARCBS) and the Australian Cord Blood Registry (AUSCORD). The donors of placentas underwent serological testing for infectious disease markers including Hep A Ab, Hep B sAg, Hep C Ab, HIV I/II Ab, HTLV I/II Ab, Syphilis Ab, EBV Ab, CMV Ab, HSV Ab, VZV Ab and Tozoplasmosis. The results had to be negative prior to placenta donation with the exception of EBV, HSV, VZV and Tozoplasmosis. A health questionnaire was carried out on the maternal donor prior to donation (using the ARCBS and AUSCORD donor guidelines) and had to comply as described earlier before the cells could be released for clinical trial use.
In addition, MSC from each passage was subjected to stringent release criteria at the time of cryopreservation and again 3 weeks prior to clinical administration. At the time of cryopreservation the following release criteria had to be met before the MSC could be released for use in clinical trials: sterility after 14 days microbiology culture; negative on mycoplasma testing, >70 % viable (using Trypan Blue exclusion); negative 16 s rRNA testing, purity defined as >85 % CD73+/CD105+ and <1 % CD45+, endotoxin testing level of <2 EU/mL and exhibition of a normal karyotype by standard cytogenetic analysis.
A schema of the production schedule is shown in Fig. 30.2.
Fig. 30.2
Human placental MSC manufacturing process
8.3 Release Criteria at the Time of Cryopreservation (After Harvest of each Passage)
In order for the cryopreserved cells to be available for subsequent use in a clinical trial, the following release criteria must be met: MSC passages (P) zero to five (P0–P5) must be sterile after 14 days microbiology culture; the MSC must be greater than 70 % viable (using Trypan Blue exclusion); MSC purity is determined by flow cytometry, and must be >85 % CD73+, >85 % CD105+ and <1 % CD45+. A normal karyotype analysis must be demonstrated on MSC from P2 to P5; mycoplasma testing (P2–P5) must be negative, 16 s rRNA testing must be negative and endotoxin testing (P2–P5) must show a level of <2 EU/mL.
9 Clinical Trials Using Mesenchymal Stromal Cells
We have completed one phase I trial of placenta-derived MSC administered intravenously in persons with idiopathic pulmonary fibrosis in conjunction with A/Professors Daniel Chambers and Peter Hopkins at Prince Charles Hospital, Brisbane. We have completed two of the three planned dose escalation cohorts with MSCs injected directly intratendinously under ultrasound guidance in persons with treatment-refractory Achilles tendinopathy in conjunction with Dr. Mark Young from Mater Private Hospital, Brisbane. There were no serious adverse events attributable to the MSCs in either trial. Both trials will be reported separately by the Principle Investigators.
10 Conclusions
In summary, we believe that the placenta represents a biologically equivalent and more readily accessible source for manufacturing human MSC for clinical trials compared to other sources. Therapeutic applications of in vitro manipulated cells represent an exciting and growing field. Cell and cell-based gene therapy manufacturing facilities need to be purpose designed and will need to be accredited by their national medicinal regulatory body. Production scientists need to work in close tandem and harmony with quality assurance and ethics committees to absolutely ensure the safety of new cellular products.
Our “open system” of manufacture is labour intensive. The optimal method of manufacturing MSC (from any source) for clinical trials would be an automated, large-scale, closed system bioreactor that could be placed in a biohazard hood and would obviate the need for costly purpose-built cGMP manufacturing suites. Such a system would be cost and time efficient and would provide optimal product safety since it would minimise the use of production personnel and the inevitable infection risk associated with multiple manual manipulations. Pluristem Therapeutics Inc. has reported on large-scale production using their proprietary PluriXTM 3D bioreactor system. This company has been able to expand adherent, placenta-derived MSC with immunomodulatory properties on a routine basis [35].
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