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Evaluation of optimal survival of primitive progenitor cells (LTC-IC) from PBPC apheresis products after overnight storage. AL Petzer, E Gunsilius, N Zech, ...
Bone Marrow Transplantation (2000) 25, 197–200  2000 Macmillan Publishers Ltd All rights reserved 0268–3369/00 $15.00 www.nature.com/bmt

Evaluation of optimal survival of primitive progenitor cells (LTC-IC) from PBPC apheresis products after overnight storage AL Petzer, E Gunsilius, N Zech, J Clausen, E Hoflehner, W Nussbaumer and G Gastl Department of Hematology and Oncology, University Hospital Innsbruck, Austria

Summary: Optimal overnight (ON) storage of PBPC aphereses is becoming an increasingly important issue and different options for storing PBPC products exist. The survival of primitive progenitor cells is of major interest, as recent data suggest that these progenitors are not only important for long-term engraftment but also contribute significantly to the early phase of hematopoietic engraftment after myeloablative therapy. We therefore investigated the survival of primitive progenitor cells (ie long-term culture initiating cells, LTC-IC) before (ie within 2 h after finishing the apheresis procedure) and after ON storage lasting 16 to 20 h. In addition, we compared the % of recovery of LTC-IC with that of mature progenitors (ie colony-forming cells, CFC) and with the % viability of the mononuclear cells in the apheresis product. Aliquots of PBPC aphereses products were tested in collection bags at room temperature (RT), in EDTA tubes both at RT or 4ⴗC ⴞ the addition of autologous plasma (AP; 2.6-fold the apheresis volume) and ⴞ the possibility of gas exchange. Mean viable cell counts did not show strong differences between the different storage conditions and were poor predictors for the survival of CFC and LTC-IC. At RT (collection bags, EDTA tubes ⴞ gas exchange) recoveries (% of input) of both, CFC (18%, 18% and 31%) and LTCIC (10%, 4%, 17%) were low. The addition of AP at RT improved the survival of CFC and LTC-IC to 66% and 38%, respectively. Optimal recoveries for both types of progenitors (CFC: 99%, LTC-IC: 109%) were obtained at 4ⴗC in the presence of AP. In addition, a good correlation between the survival of CFC and LTCIC was obtained (r = 0.76) suggesting that the analysis of CFC may also allow some conclusions to be drawn on the survival of LTC-IC. Bone Marrow Transplantation (2000) 25, 197–200. Keywords: overnight storage; stem cells; LTC-IC; progenitor cells; colony-forming cells; survival

the day, on Sundays or holidays without the need to process the apheresed cells late in the evening or on holidays, respectively. In addition, further in vivo purging procedures such as positive selection for CD34-positive cells or negative selection techniques using immunoaffinity columns or immunomagnetic devices could be better coordinated and optimized. Recommendations concerning this issue exist but vary among the different centers and are primarily based on analyses of viable cell counts, measurement of CD34-positive cells and/or colony-forming cells (CFC) after ON storage.1–5 In addition, many of these analyses were performed on bone marrow progenitors and the conclusions drawn from these experiments were translated to PBPC without re-evaluation. Recent animal data suggest that primitive progenitor cells are not only important for long-term engraftment but also contribute significantly to the early phase of hematopoietic engraftment after myeloablative therapy.6,7 Moreover, transplantation of higher numbers of rhodamine-negative hematopoietic stem cells has resulted in an accelerated reconstitution.7 These animal data seem to be relevant also in humans. When patients with multiple myeloma or breast cancer were transplanted with only a small subpopulation of CD34+ cells, that is highly enriched for primitive stem cells (ie CD34+Thy1+), a rapid hematologic engraftment of all lineages occurred.8,9 Primitive progenitors should therefore be the main target to be analyzed after ON storage. Cells identified as long-term culture initiating cells (LTC-IC) have been shown to overlap with cells having in vivo repopulating activity (referred to as competitive repopulating units or CRU)10,11 and might therefore serve as a surrogate marker for the specific quantitation of these cells. We were therefore primarily interested in the analysis of optimal recoveries of primitive progenitor cells (LTC-IC) using different ON storage conditions and to see whether correlations with the survival of mature progenitors (CFC) and the viability of the cells could be found.

Materials and methods Cells

Overnight (ON) storage of mobilized peripheral blood progenitor cells (PBPC) is becoming increasingly important for various reasons. This would allow PBPC harvesting late in Correspondence: Dr AL Petzer, Universita¨tsklinik Innsbruck, Abteilung fu¨r Ha¨matologie und Onkologie, Anichstr. 35, A–6020 Innsbruck, Austria Received 13 May 1999; accepted 25 August 1999

Aliquots (100 ␮l) of mobilized cells were collected from patients with non-Hodgkin’s lymphoma (n = 4), Hodgkin’s disease (n = 1), multiple myeloma (n = 1), acute myeloid leukemia (n = 2), medulloblastoma (n = 1) or breast cancer (n = 3) after obtaining informed consent. Following chemotherapy and subsequent treatment with filgrastim (G-CSF, Neupogen, Amgen, Vienna, Austria), apheresed cells were

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Figure 1 Mean viable cell counts (Trypan blue dye exclusion test) ⫾ s.e.m. of apheresed MNCs (n = 12) before and after different ON storage conditions. *Indicates statistical significance (P ⬍ 0.05) to MNC analyzed immediately after the apheresis procedure.

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Storage conditions Figure 3 Mean % recovery ⫾ s.e.m. of LTC-IC (n = 7) after different ON storage conditions (compared to input LTC-IC analyzed within 2 h after finishing the apheresis procedure). *Indicates statistical significance (P ⬍ 0.05) to all other conditions tested.

and 3). In parallel experiments, autologous plasma (AP) was added at 2.6-fold the original volume and/or the lid of the tubes was loosened to allow the exchange of gas during the storage.

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Analysis of cell viability and the survival of CFC and LTC-IC

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Cell viability was determined before (ie within 2 h after finishing the apheresis procedure) and after ON storage using the Trypan blue dye exclusion test. Aliquots of apheresed cells were assayed for committed progenitor cells (colony-forming cells, CFC) as described.12 In brief, erythropoietic burst-forming unit (BFU-E), granulocyte–macrophage (CFU-GM) and mixed (CFU-granulocyte–erythroid–macrophage–megakaryocyte CFU-GEMM) CFC were enumerated by plating cells at suitable concentrations (to provide ⬍100 colonies per 1 ml culture) in Iscove’s medium containing 0.9% methylcellulose, 30% FCS, 1% BSA, 10−4 m 2–mercaptoethanol (2-ME), (Methocult H 4430; StemCell Technologies, Vancouver, BC, Canada) supplemented with 3 U/ml of highly purified human erythropoietin (Erypo, Janssen-Cilag, Austria), 50 ng/ml of stem cell factor (Amgen, Vienna, Austria), and 20 ng/ml each of interleukin (IL)-3 (Sandoz International, Basel, Switzerland), IL-6 (R&D, Biomedica, Vienna, Austria), granulocyte colony-stimulating factor (G-CSF, filgrastim, Neupogen, Amgen), and granulocyte–macrophage colony-stimulating factor (GM-CSF, molgramostim, Leucomax; Aesca, Traiskirchen, Austria). Methylcellulose cultures were aliquoted in 1.1 ml volume in 35-mm Petri dishes (Greiner, CA & So¨hne, Labortechnik, Kremsmu¨nster, Austria) and then incubated at 37°C for 2–3 weeks. All colonies present were scored according to standard criteria.12 For enumeration of LTC-IC, cells to be tested were placed in 35-mm tissue culture dishes that already contained a feeder layer of irradiated (8000 cGy) mouse fibroblasts (M210B4) plus 2.5 ml of human long-term culture medium (HLTM).13 HLTM (MyeloCult H 5100; StemCell Technologies) contained 12.5% horse serum, 12.5% FCS, 10−4 m 2-ME and 10−6 m hydrocortisone sodium hemisuccinat (Sigma H 4881, Vienna, Austria) which was added from a freshly prepared solution just before use. The cultures were then incubated for 6 weeks at 37°C with weekly

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Figure 2 Mean % recovery ⫾ s.e.m. of CFC (n = 7) after different ON storage conditions (compared to input CFC analyzed within 2 h after finishing the apheresis procedure). *Indicates statistical significance (P ⬍ 0.05) to bag and EDTA tube without the possibility of GE and the addition of AP at RT. †Indicates statistical significance (P ⬍ 0.05) to EDTA tube + GE at RT.

analyzed within 2 h after finishing the apheresis procedure. PBPC collections were performed on a Cobe Spectra (COBE, Lakewood, CO, USA; n = 4) or on a CS 3000 separator (Baxter, Deerfield, IL, USA; n = 8). Cells were either left in the collection bag (from the disposable set, number 777–006–000; COBE, or from the open system apheresis kit, BL 146; Baxter) or transferred into sterile EDTA tubes (Monovette, Sarstedt; Nu¨mbrecht, Germany) and stored ON for 14 to 18 h under different conditions as described. Cell densities varied from 2.2 × 108 to 7.6 × 108 cells/␮l. Storage conditions Cells were either stored ON in the collection bag at room temperature (RT, 20°C) or in EDTA-coated tubes, both at RT and at 4°C. Initial experiments had shown that the survival of CFC and LTC-IC did not show significant differences between these two conditions at RT (data not shown). Furthermore, retrospective analyses of the experiments performed here again showed no significant difference in cell viability, survival of CFC and LTC-IC between cells left in the collection bag or transferred to the tube (Figures 1, 2 Bone Marrow Transplantation

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replacement of half of the medium and removal of half of the nonadherent cells.13,14 After 6 weeks, all of the remaining nonadherent cells from each assay were pooled with the corresponding adherent cells, washed and assayed for CFC as described.12–14 The counts obtained were used to calculate the total yield of CFC (BFU-E plus CFU-GM plus CFU-GEMM) at the end of 6 weeks from each input inoculum tested as this value provides a relative, but nevertheless quantitative measure of the LTC-IC initially present.12,13 The numbers of CFC and LTC-IC were analyzed from cultures initiated within 2 h after finishing the apheresis procedure and defined as a value of 100%. The numbers of progenitors analyzed after ON storage were expressed as percentage of this value.

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% LTC-IC recovery Figure 4 Correlation of matched pairs of % CFC and % LTC-IC recoveries analyzed after different ON storage conditions.

Statistical analysis Differences between the mean values of the various storage conditions were analyzed using the Mann–Whitney test from the GraphPad InStat software program (PRISM Version 3.0 for Windows 95). Correlations were tested by linear regression analyses.

Results Cell viability The most common method for analyzing the quality of apheresis products is the use of the Trypan blue dye exclusion test. As can be seen in Figure 1, mean viability values for all different storage conditions were ⭓74% and no strong differences between the various conditions tested were found. The lowest viability values were found when the cells were stored at RT (bag or tube) without further manipulations such as the possibility of gas exchange (GE) or the addition of AP. The mean viability values for these ON storage conditions were significantly worse compared to apheresed cells analyzed immediately after the apheresis procedure (P ⬍ 0.05, Figure 1). Survival and recovery of comitted progenitor cells (CFC) In contrast to viability cell counts, the survival of CFC showed strong differences between the different storage conditions. As shown in Figure 2, only 17.9 ⫾ 4.4% (mean ⫾ s.e.m.) and 18.0 ⫾ 6.4% of the CFC recovered when the cells were stored at RT in the bag or in the tube. The possibility of gas exchange slightly increased the survival of CFC to 31.2 ⫾ 10.8%. The addition of AP was able to further improve the recovery of CFC to 66.0 ⫾ 17%. A similar recovery of 68.9 ⫾ 14.4% was obtained when cells were kept in the tube at 4°C with the possibility of GE. An optimal recovery (99.9 ⫾ 8.5%) was observed when the cells were kept at 4°C with the possibility of GE plus the addition of AP. Both storage conditions at 4°C were significantly better (P ⬍ 0.05) than the bag or the tube at RT without the possibility of GE or the addition of AP.

Survival and recovery of primitive progenitor cells (LTC-IC) Overall, LTC-IC survived better at 4°C compared to RT (Figure 3) and again recovered best (109.4 ⫾ 12.4%) under conditions that also allowed optimal survival of CFC (ie 4°C, GE and AP). Storage of PBPC at 4°C with the possibility of GE and the addition of AP was significantly superior (P ⬍ 0.05) to all other conditions tested. Interestingly, the survival of CFC and LTC-IC was similar under the different storage conditions. Figure 4 shows that a good correlation between these two types of progenitors was obtained (r = 0.76). In contrast, viable cell counts only poorly predicted the survival of both types of progenitor cells with correlation coefficients of 0.54 (CFC vs cell viability) and 0.49 (LTC-IC vs cell viability), respectively. Discussion Many studies on the short-term storage of hematopoietic progenitor cells for transplantation have reported data on bone marrow cells and suggest that storage of the cells at RT as well as 4°C is feasible. To our knowledge, no previous reports have analyzed the survival of primitive progenitor cells (and putative stem cells) – including more recent reports on liquid storage of PBPC obtained by apheresis. In a recent publication by Pettengell et al15 the capacity of progenitor cells to sustain hematopoiesis up to 3 weeks in LTC was analyzed after 72 and 120 h, respectively. It has to be noted, however, that only cells that are capable of generating CFC for at least 5 weeks in LTC meet the criterion for LTC-IC that overlap with potential stem cells (CRU).10,11,16 Our data clearly show that for LTC-IC significant differences exist between the two types of temperatures (ie RT vs 4°C). In general, storage at 4°C was always superior to the storage at RT under otherwise identical conditions in terms of the survival of both committed (CFC) and primitive progenitor cells (LTC-IC). In addition, parameters such as the possibility of gas exchange and, more important, the addition of AP seem to be beneficial. This may be due to the fact that the addition of AP increases the total volume 2.6-fold and consequently cell densities decrease. A recent report has shown that high cell densities are associated with significant losses of CFC after liquid storage.17 In addition, (growth) factors in the plasma and less dramatic Bone Marrow Transplantation

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changes in pH values may also support the survival of the progenitor cells. It is important to note that the analysis of cell viability only poorly predicts the survival of both types of progenitors, CFC and LTC-IC, respectively. In contrast, a good correlation was found between the survival of CFC and LTC-IC (r = 0.76) after ON storage, suggesting that the evaluation of CFC (much easier to perform) might be a valid parameter for the analysis of the quality of the PBPC collections after ON storage which will allow some conclusions to be drawn on the survival of the more primitive progenitors. Optimal survival of progenitor cells after ON storage is important since it has been reported that, in general, the number of progenitor cells per kg reinfused after myeloablative chemotherapy correlates with the time to neutrophil and platelet recovery.18,19 In this context, primitive progenitors become more and more important because there is evidence that higher numbers of primitive cells may result in an accelerated hematopoietic reconstitution.7 Our data also suggest that PBSC collections from unrelated donors (which probably will be performed more frequently over the next few years) should be transported at 4°C. To date, recommendations in this respect by the World Marrow Donor Association only exist for the transportation of bone marrow.20 In order to limit costs and labor for CD34+ enrichment procedures using immunoaffinity or immunomagnetic devices, combining fresh and ON stored leukapheresis products is a commonly employed procedure in many centers. The recovery and purity of CD34+ cells after the CD34+ enrichment procedure is known to be dependent on the percentage of viable cells before this procedure.5 Therefore, an optimum of ON storage conditions, as described here, should be aimed at.

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The authors want to thank P Schumacher for excellent technical assistance, Amgen and Janssen-Cilag Austria for generous gifts of growth factors used to analyze CFC and LTC-IC and M Bezdekowsky from Baxter Austria for the discussion of storage conditions.

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