CD34 Positive Selection of Cryopreserved Stem Cell Concentrates with the CliniMACS Prodigy Platform and the Tubing Set TS 320: Preclinical Results from a Validation Study

Abstract

Introduction

Immuno-magnetic CD34 positive (CD34+) selection is generally used for stem cell boosts after allogeneic stem cell transplantation. In some cases, only cryopreserved cells are available as starting material. We present a new automatic device and technique for preparing and subsequently magnetically enriching these cells.

Methods

We used the CliniMACS Prodigy® platform in combination with the TS 320 tubing set, equipped with a large 800 mL chamber, which enables to include the DMSO wash, platelet wash, antibody incubation, and antibody wash and the subsequent CD34 enrichment on an automatic basis with only short hands-on time. We performed three validation runs using DNase and sodium citrate for cell preparation to reduce the risk of aggregates and clots.

Results

Our robust preclinical results show the feasibility and safety of the process with a mean of 53% CD34 yield after thawing and CD34 purity and viability of 93 and 97%, respectively. CD3 log depletion exceeds 5.0 in all 3 cases, which provides optimal GvHD prevention.

Conclusion

This shows that the updated technique gains unaffected CD34+ cells of high quality, not only from fresh but also from cryopreserved stem cell products.

Introduction

Immuno-magnetic CD34 positive (CD34+) selection is a graft manipulation technique to lower the risk of graft-versus-host disease in patients receiving stem cell boosts for poor graft function after allogeneic hematopoietic stem cell transplantation. In the last years, this has been done using the CliniMACS Plus^® device (Miltenyi Biotec). The new device, CliniMACS Prodigy^®, in combination with the tubing set TS 320, equipped with a large 800 mL chamber, enables a large-scale CD34+ enrichment. In contrast to the CliniMACS Plus, the process on the Prodigy platform includes the DMSO wash, platelet wash, antibody incubation, and antibody wash. We report the preclinical data of the successful validation of the CD34+ selection of hematopoietic stem cells out of cryopreserved stem cell products using the CliniMACS Prodigy^® with the tubing set TS 320.

Methods

Three cryopreserved allogeneic peripheral stem cell grafts from deceased recipients (with informed consent of the donors) were selected for this validation process after storage in the vapor phase of liquid nitrogen for 1.5, 1.7, and 5.5 years, respectively. Detailed characteristics of the stem cell products at the time of donation are presented in Table 1.

Table 1.

Characteristics of basic stem cell products

​	Fresh product		Cryopreserved product post-donation
	CD34+ cells, n × 106	WBC, n × 109/L	MNC %	PLT, n × 1011	CD34+ cell viability, %	WBC, n × 109/L
Run 1	245	186	80	1.7	84	186
Run 2	214	228	75	1.2	95	228
Run 3	290	138	76	0.7	92	136

The stem cells were thawed carefully at 37°C for 5 min in the original cryo bag in a dry heating device (Plasmatherm DTM V201, Barkey) until only some ice crystals were visible and immediately (<1 min) supplemented with 2.5 mL/20 mL DNase (Pulmozyme^® 2,500 E/2.5 mL). DNase was used to avoid clumping of cells caused by cell-free DNA. A sample for flow cytometry was taken.

Next, the product was diluted 1:5 with dilution buffer (MACS^® GMP Tyto^® running buffer with magnesium chloride, supplemented with 1% human albumin and 0.4% sodium citrate [100 mL buffer+ 13.3 mL sodium citrate 30%]) in a 600 mL transfer bag. The absence of EDTA in the buffer is crucial to avoid inactivation of magnesium chloride, which is necessary for the function of DNase. A minimum 1:3 dilution is recommended to reduce leucocyte and platelet concentration in the starting product.

According to the protocol, all reagents were connected to the Prodigy device. Overall, 6 L running buffer consisting of MACS^® GMP Tyto^® Running Buffer with magnesium chloride supplemented with 1% human albumin and 10 mL DNase were interconnected and placed on the Prodigy device. Next, one vial CliniMACS CD34 Reagent and one vial immunoglobulin were transferred to the designated reagent bags on the tubing set. Finally, the elution buffer consisting of 0.9% sodium chloride solution supplemented with 1% HSA was welded to the tubing set.

After connection to the Prodigy tubing set, the cells were transferred to the application bag including a filtration step to reduce possible aggregates. After that, the enrichment process was initiated.

First, cells were washed to reduce DMSO and platelets to a minimum. In the next step, the cells were incubated with immunoglobulin and labelled with anti-CD34 immune-magnetic beads. Subsequently, washing steps were performed to reduce the unbound antibody. The magnetic enrichment and the elution were automated without further practical work.

The entire process took 5 h, including 2 h of hands-on time. After the run, the target cell bag, the non-target cell bag, and the waste bags were sealed off and samples were taken for quality controls.

Results

Flow cytometry analyses for CD45+ FITC, CD34+ PE (Stem-Kit reagents), and CD3+ PC5 cells (Cyto-Stat Tetrachrome), as well as viability analyses using 7-AAD, all from Beckman Coulter, were performed on a single-platform basis (Navios 10 colors, Beckman Coulter). Sterility was tested by bacterial culture (BACT/ALERT^®, bioMerieux).

Prior to cryopreservation, the mean basic value of viable CD34+ cells was 250 ± 38 × 106. Cryopreserved material of the respective three stem cell concentrates was used for the CliniMACS Prodigy^® validation runs. Thawing of the cells lead to a mean loss of 24 ± 6%, resulting in 191 ± 42 × 10⁶ CD34+ at the start of the cell preparation. All three materials were successfully processed and enriched for CD34+ cells. The mean end volume was 84 ± 4 mL comprising 101 ± 18 × 10⁶ viable CD34+ cells (mean yield 53 ± 4%), while the mean CD3+ log depletion of −5.2 was also excellent. No clots or aggregates were detected during the whole process. Sterility testing was negative for all samples. The detailed results of the three runs are presented in Table 2.

Table 2.

Results from three validation runs

​	Thawed product	Final cell product post-enrichment
	viable CD34+ cells, n × 106	viable CD34+ cells, n × 106	CD34+ cell yield, %	CD34+ cell purity, %	CD34 + cell viability, %	CD3+ cell log depletion
Run 1	189	107	57	94	97	−5.1
Run 2	150	81	54	91	99	−5.0
Run 3	234	115	49	94	96	−5.4

Discussion

The administration of stem cell boosts with CD34+ selected stem cell products without conditioning is utilized for poor graft function after allogeneic stem cell transplantation or CAR-T cell therapy [1, 2]. Until now, this has been achieved using the CliniMACS Plus device in-house. As in other laboratories, the Prodigy platform has been successfully implemented for the CD34+ selection of fresh stem cell grafts [3–5]. Unfortunately, fresh stem cell products are not always available, so cryopreserved starting materials are used instead. Consistent with our experience of using the CliniMACS Plus device (n = 6), Ghobadi et al. [6] have reported that selecting previously cryopreserved cells yields comparable clinical outcomes to those achieved with fresh cells. However, in this case, a separate cell wash is required before the CD34+ enrichment process to remove DMSO. Furthermore, despite adding DNase and ACD-A, massive clotting of the thawed apheresis product occurred, which made the process extremely difficult. Nevertheless, with this process from cryopreserved material on the CliniMACS Plus device, we achieved mean ± SD values of 95 ± 4% pure and 94 ± 7% viable CD34+ cells and a mean recovery of 51 ± 5% compared to basic values.

Our recent results show the feasibility and safety of CD34+ selection of cryopreserved stem cell grafts with the Prodigy platform. With an average CD34 yield of 53 ± 4%, our results are comparable to internal data of the manufacturer (yield of 52 ± 9% [n = 15] with and 66 ± 6% [n = 12] without overnight storage from fresh apheresis products). Moreover, with the CliniMACS Prodigy, the high purity and viability of the CD34+ cells and the absence of aggregates or clots during the graft manipulation procedure was achieved by the immediate >1:3 dilution of the thawed product with an optimized DNase-supplemented buffer. Furthermore, DMSO was washed out thoroughly within a short time guaranteed by the automated Prodigy process with the large chamber. These aspects provide a robust strategy for successful clinical results using cryopreserved material for stem cell boosts.

To our knowledge, we are the first to report the results of using the Prodigy platform and the tubing set TS 320 to select CD34+ cells from cryopreserved stem cell grafts, incorporating DMSO removal directly into the automated enrichment process. The low number of validation runs limits our work; nevertheless, the variability of the results is low, and improving our skills might further enhance the outcome.

In summary, the results of the CD34+ yield, purity, and viability are satisfactory and sufficient for a stem cell boost. The efficient T-cell reduction in particular provides optimal graft-versus-host disease prevention.

Acknowledgments

Especially, we thank Heike Lahnor, Katharina Krämer, Amina Dikme, and Hans-Dieter Steibl, Miltenyi Biotec and Miltenyi Biomedicine, respectively, for their support in the practical process.

Statement of Ethics

The study was reviewed and approved by the Ethics Committee of the Medical University of Graz. Written informed consent was obtained from the stem cell donors prior to donation.

Conflict of Interest Statement

The authors declare no conflicts of interest. Peter Schlenke was a member of the journal’s Editorial Board at the time of submission.

Funding Sources

Reagents and tubing sets were supplied by Miltenyi Biotec. There was no other financial funding.

Author Contributions

P.S., K.R., and C.B. contributed in the conception and design of the study. K.R., C.U., A.R., and C.B. performed the hands-on work in the clean room and data acquisition. K.R. and C.B. conducted the data interpretation and writing of the manuscript. P.S. contributed with discussion and critical revision of the manuscript.

Funding Statement

Reagents and tubing sets were supplied by Miltenyi Biotec. There was no other financial funding.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.