Small volume bone marrow aspirates with high progenitor cell concentrations maximize cell therapy dose manufacture and substantially reduce donor hemoglobin loss

DKMS [16, 17] is a major international stem cell donor center with 12 million registered donors in 7 countries (Chile, Germany, India, Poland, South Africa, the UK, the USA). Since 1991, donors registered with DKMS have donated stem cells (BM or PBSC) 111,000 times, including 7705 times in 2022. In that year, DKMS donors contributed 35.4% (7705/21,767) of all unrelated stem cell products worldwide [18]. DKMS receives an individual collection report following each stem cell collection of a DKMS donor. This report includes TNC count and concentration, and the ratio of collected to requested TNCs. Reports were analyzed for BM collections performed in Germany (GER; N = 910, of which 81 were performed at our center), Poland (PL; N = 209), the USA (USA; N = 96), and the UK (UK; N = 33) in 2019. Overall, 25 collections were excluded from the analysis (21 from GER, 2 from UK, 1 from PL, 1 from USA) since relevant information was missing on the collection report.

The study was approved by the ethics committee of the Goethe University Hospital, Frankfurt, Germany (approval #19–286).

Human BM samples were obtained after informed donor consent from 20 healthy, adult allogeneic donors (4 female and 16 male, aged 18–56 [mean: 29.6] years; Table 1) during BM collections for clinical HPC-M products using a Jamshidi needle (SELECTIVE “ILY” type, ZAMAR®, Vrsar, Croatia). Small volume (3 mL) and large volume (10 mL) aspirates, anticoagulated with heparin (Li-Heparin Vacutainer, Becton–Dickinson, Heidelberg, Germany), were sampled at the start (t1), at halftime (t2), and at the end (t3) of each BM collection. To standardize sampling and to exclude possible impact of the needle position in the punction canal, each sample was taken from separate punctions as first aspirate after reaching the BM cavity. In detail, a 3-mL BM aspirate was sampled immediately (first aspirate) after the first puncture of the iliac crest at t1 (puncture A). The following aspirates of this puncture channel were used for the HPC-M product. In a second, independent, puncture location at t1, a 10-mL BM aspirate was collected as first aspirate of this channel (puncture B), and the following aspirates of this channel were used for the product. This was repeated at t2 (punctures C and D) and at t3 (punctures E and F) (Additional file 1: Fig. S1). To minimize possible operator effects, six operators in total performed BM sampling. Thereby, different individuals took the small and large volume samples randomly. For calculation of cell concentrations, exact volumes of the BM samples were recorded.

After dilution with DPBS (Thermo Fisher Scientific, Darmstadt, Germany) and filtering (CellStrainer, Corning, Amsterdam, The Netherlands), TNC counts, hemoglobin content, hematocrit, RBC, and platelet counts were determined using the Sysmex XN-350 automatic hemocytometer (Sysmex Deutschland GmbH, Norderstedt, Germany). The BM aspirates were further processed by density gradient centrifugation on lymphocyte separation medium (Lonza, Basel, Switzerland). Cell pellets were resuspended in DPBS and TNC counts determined using Sysmex XN-350.

Flow cytometry was performed with LSRFortessa (Becton–Dickinson). In detail, 2 × 10⁶ cells each were stained in three different multicolor panels, comprised of markers for HSPCs and endothelial progenitor cells (EPCs), immune cell subsets and MSCs and their precursors, followed by RBC lysis (BD Pharm Lyse™ Lysing Buffer, BD Biosciences, Heidelberg, Germany).

For detection and quantification of HSPCs and EPCs, cells were stained with 7-AAD viability dye (BioLegend, San Diego, CA, USA) and the following antibodies (all from BioLegend, unless otherwise noted): anti-CD14-APC-Cy7 (63D3), anti-CD31-Brilliant Violet 605™ (WM59), anti-CD34-Alexa Fluor® 700 (581), anti-CD45-V500 (HI30, BD Horizon™), anti-CD117-Alexa Fluor® 488 (104D2), anti-CD133-Brilliant Violet 421™ (clone 7), and anti-CD309-PE (7D4-6).

For analysis of immune cell subsets, cells were stained with 7-AAD viability dye and the following antibodies (all from BioLegend): anti-CD3-Pacific Blue (HIT3a), anti-CD4-Alexa Fluor® 700 (SK3), anti-CD8-Brilliant Violet 510™ (SK1), anti-CD11b-PE-Cy7 (LM2), anti-CD14-APC-Cy7 (63D3), anti-CD19-PE (HIB19), anti-CD45-FITC (HI30), anti-CD56-Alexa Fluor® 647 (5.1H11), anti-CD183-Brilliant Violet 605™ (G025H7), and anti-CD194-PE-Dazzle™ 594 (L291H4).

MSCs and their precursors were detected in the BM aspirates by staining with 7-AAD viability dye and the following antibodies (all from BioLegend, unless otherwise noted): anti-CD29-APC-Cy7 (TS2/16), anti-CD34-Alexa Fluor® 700 (581), anti-CD45-FITC (HI30), anti-CD73-Brilliant Violet 605™ (AD2), anti-CD90-PE-Cy7 (5E10, BD Horizon™), anti-CD105-PE-CF594 (266, BD Horizon™), anti-CD119-PE (GIR-208), anti-CD146-Brilliant Violet 510™ (P1H12), and anti-CD271-BV421 (C40-1457, BD Horizon™).

Flow cytometry data were analyzed using FCS Express 6 Flow Software (De Novo Software, Pasadena, CA, USA). The percentages of viable cells of each cell type were converted to cell count per milliliter processed BM.

To quantify HSPCs on functional level, the numbers of colony-forming unit cells (CFU-C) were assessed. Briefly, 2.5 × 10⁴ cells per replicate were resuspended in Iscove’s Modified Dulbecco’s Medium (IMDM) (Thermo Fisher Scientific) with 2% fetal bovine serum (FBS) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and plated in duplicates into methylcellulose-based medium (MethoCult H4434 Classic; Stemcell Technologies SARL, Saint Égrève, France) in 35 mm cell culture dishes. Cultures were maintained at 37 °C in humidified atmosphere with 5% CO₂ for 10–16 days. Colonies were counted and evaluated by inverted microscopy with tenfold magnification, thereby differentiating burst-forming unit-erythroid (BFU-E), colony-forming unit-macrophage (CFU-M), colony-forming unit-granulocyte, macrophage (CFU-GM), and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM). Using the known sample volume, the input cell count and the TNC, the number of colonies counted was converted to CFU-C per milliliter of processed BM applying the rule of three.

To determine the MSC progenitor content, the numbers of colony-forming unit fibroblasts (CFU-F) were assessed. In detail, 7.5 × 10⁵ cells per replicate were resuspended in standard cell culture medium, composed of Alpha MEM (Lonza), 10% human platelet lysate (hPL) (manufactured in-house), 2 IU/mL heparin (Ratiopharm, Ulm, Germany), and 1% penicillin–streptomycin (Thermo Fisher Scientific) and seeded in duplicates in 2 wells of a 6-well plate. Cultures were maintained at 37 °C in humidified atmosphere with 5% CO₂ for 7–8 days. CFU-F were stained with 0.5% crystal violet solution (Sigma-Aldrich) and counted.

Using the known sample volume, the input cell count and the TNC, the number of colonies counted was converted to CFU-F per milliliter of processed BM applying the rule of three.

The required BM volume for a given HPC-M product was projected using the formulaeand

The projected required BM volume was then used to calculate the respective expected Hb loss.

The number of MSC doses at passage (P) 3 that can be manufactured from a given BM volume was calculated as follows.

Based on the flow cytometry data presented here, up to 0.1% of the TNCs are MSC precursors. Thus, multiplying 0.1% of TNC per milliliter processed BM by the given BM volume gives the maximum number of MSCs that can be obtained at the end of P0 (= N₀).

For the following passages, the projected MSC yield was calculated according to the formula N = N₀ × 2ⁿ, where N is the projected MSC yield at end of the respective passage, N₀ is the projected MSC yield at P0, and n are the cumulative population doublings (cPDs) of the respective passage, i.e., number of cumulative doublings occurring in this passage. For the calculation, we used representative cPDs for MSCs as reported previously [19].

Quantitative data are presented as means ± standard error of the mean (SEM) unless stated otherwise and were, following testing for normal distribution, compared with two-tailed t-tests (Fig. 2), Wilcoxon matched pairs tests (Figs. 3, 4, 5, 6, and 7B, D, Supplementary Figs. 4-6A, 7–9), analyses of variance (ANOVA) (Fig. 1, Supplementary Figs. 2, 6B/C), and post hoc tests as specified in the figures using GraphPad Prism (La Jolla, CA, USA). Exact p-values are reported in the figures, and p < 0.05 was considered as statistically significant.

For each sampling time (t1, t2, t3), we calculated the quotients of concentration of a cell type in small to large volume aspirates. With these ratios, we performed non-parametric Spearman correlation analysis (GraphPad Prism) for TNCs versus cell population of interest to assess whether the enrichment of various cell types in small volume aspirates is solely due to the increased content of TNCs.

To visualize the relationship of a cell population’s ratio (small/large) to TNC ratio (small/large), we performed linear regression (GraphPad Prism) and showed a perfect fit line (TNCs) for comparison, when TNCs alone would explain the enrichment of different cell populations in small volume aspirates.

The cartoon (Supplementary Fig. 3) was created with BioRender.

When we compared HPC-M products collected at our center to those collected in other centers worldwide for DKMS donors in 2019, we found that the TNC contents varied substantially. Specifically, the HPC-M products that were collected in our center had higher TNC concentrations (average of 25.01 × 10⁶/mL) and higher TNC counts (average of 22.89 × 10⁹ TNCs per product) when compared to other international centers (Fig. 1, Additional file 1: Fig. S2A). Such, we met, or even exceeded, the requested TNC numbers in more cases than other centers (Additional file 1: Fig. S2B). For several years, we have implemented a standard technique for BM collections allowing a maximal volume of 5 mL for each individual BM aspirate, whereas other centers may manufacture the HPC-M products with larger volume BM aspirates. This prompted us to test our hypothesis that small volume BM aspirates are higher concentrated for nucleated cells, whereas the larger BM aspirate volumes contain more RBCs per milliliter, thus collecting fewer nucleated cells per volume unit (Additional file 1: Fig. S3).

First, we analyzed the concentrations of TNCs per mL BM in small volume aspirates and large volume aspirates. We found that each milliliter of small volume aspirates contained significantly more TNCs than large volume aspirates (average of 4.62 × 10⁷ [small] vs. 3.04 × 10⁷ [large]; p = 0.0001) (Fig. 2A). Accordingly, the concentration of CD45 + cells was higher in the small volume aspirates (average of 4.0 × 10⁷/mL [small] vs. 2.62 × 10⁷/mL [large]; p = 0.0001) (Fig. 2B). Next, we aimed to investigate the immune cell composition of the BM and if we would find differences between the small and large volume aspirates. Therefore, we analyzed the CD45 + population for immune cell subsets and identified neutrophils as most frequent immune cell types followed by T cells in small and large volume aspirates. Both neutrophils (average of 2.64 × 10⁷/mL [small] vs. 1.74 × 10⁷/mL [large]; p = 0.0003) and T cells (average of 5.14 × 10⁶/mL [small] vs. 3.75 × 10⁶/mL [large]; p = 0.0005) were higher concentrated in small volume aspirates (Additional file 1: Fig. S4A; Fig. 3). Further characterizing the immune cell subsets by multicolor flow cytometry, we found that B cells (average of 2.67 × 10⁶/mL [small] vs. 1.59 × 10⁶/mL [large]; p = 0.0003), NK cells (average of 1.68 × 10⁶/mL [small] vs. 0.97 × 10⁶/mL [large]; p = 0.0001), monocytes/macrophages (average of 1.44 × 10⁶/mL [small] vs. 0.82 × 10⁶/mL [large]; p = 0.0002), cytotoxic T cells (CTLs) (average of 2.1 × 10⁶/mL [small] vs. 1.43 × 10⁶/mL [large]; p = 0.0002), NK-T cells (average of 1.75 × 10⁵/mL [small] vs. 1.27 × 10⁵/mL [large]; p = 0.0002), and suppressor T cells (average of 2.08 × 10⁴/mL [small] vs. 1.65 × 10⁴/mL [large]; p = 0.0033) were significantly higher concentrated in small volume aspirates compared to large volume aspirates (Additional file 1: Fig. S5; Fig. 3). We also observed higher concentrations of T Helper (Th) cells in small volume aspirates (average of 2.73 × 10⁶/mL [small] vs. 2.07 × 10⁶/mL [large]; p = 0.0006). Interestingly, the concentration effect in the small volume aspirate pertained only to Th₂ cells (average of 5.24 × 10⁵/mL [small] vs. 4.21 × 10⁵/mL [large]; p = 0.0004), whereas Th₁ cells trended higher in large volume aspirates (average of 0.93 × 10⁵/mL [small] vs. 1.02 × 10⁵/mL [large]; p = 0.4024 (Fig. 3). In addition, the platelet concentrations were higher in the small volume aspirates compared to the large volume aspirates (average of 1.53 × 10⁸/mL [small] vs. 1.07 × 10⁸/mL [large]; p = 0.0003) (Additional file 1: Fig. S4B).

We showed that, compared to large BM volume aspirates, small volume aspirates concentrate TNCs, most immune cell types and platelets. Therefore, we tested if HSPCs would also follow this principle. Identified by multicolor flow cytometry, we found substantially more CD34pos (average of 7.45 × 10⁵ [small] vs. 4.26 × 10⁵ [large]; p < 0.0001), CD133pos (average of 5.18 × 10⁵ [small] vs. 3.24 × 10⁵ [large]; p = 0.0004), and CD34posCD133pos (average of 3.93 × 10⁵ [small] vs. 2.41 × 10⁵ [large]; p < 0.0001) cells per milliliter in small volume aspirates (Fig. 4).

To confirm the stem/progenitor character of the cells, we additionally quantified the hematopoietic colony forming HSPC clones. In line with the multicolor flow cytometry results, small volume BM aspirates had higher concentrations of BFU-E (average of 6.34 × 10⁴/mL [small] vs. 3.66 × 10⁴/mL [large]; p < 0.0001), CFU-M (average of 1.04 × 10⁵/mL [small] vs. 0.59 × 10⁵/mL [large]; p < 0.0001), CFU-GM (average of 2.21 × 10⁴/mL [small] vs. 1.02 × 10⁴/mL [large]; p = 0.0001), and CFU-GEMM (average of 9.11 × 10²/mL [small] vs. 3.52 × 10²/mL [large]; p = 0.0033) (Fig. 5). The concentrations of HSPCs in both small volume and large volume BM aspirates decreased over time suggesting a kinetic of HSPC harvest during the individual collection process. Yet, the reduction of HSPC concentration was significant only for large volume aspirates, and the small volume aspirates contained still significantly more HSPCs per milliliter at each time point during the individual BM collections (Additional file 1: Fig. S6).

The majority of the tested cell types were higher concentrated in the small volume aspirate; however, this was not the case for all cell types. Thus, we assessed, as a next step, the concentration effect of small volume aspirates for additional, non-hematopoietic progenitor cells types that have been increasingly used for cell therapies manufacture, i.e., MSCs and EPCs. In the human BM, MSC populations and their precursors can be found in the CD45neg and CD45dim fractions [20, 21]. Both were higher concentrated in the small volume aspirates (CD45neg: average of 5.52 × 10⁶/mL [small] vs. 4.04 × 10⁶/mL [large]; p = 0.0048; CD45dim: average of 6.79 × 10⁵/mL [small] vs. 4.69 × 10⁵/mL [large]; p = 0.0001) (Fig. 6A, B). As MSCs feature substantial heterogeneity, we quantified the MSC subpopulations with multicolor flow cytometry and found for both CD45neg and CD45dim cells the CD146posCD29pos, CD146posCD119pos, and CD271posCD90pos as most prevalent subpopulation phenotypes. We detected all MSC subpopulations at higher concentrations in the small volume aspirates (Additional file 1: Fig. S7 + 8). Quantifying the MSC progenitors by CFU-F, we confirmed the concentration effect of the small volume aspirates for MSCs (average of 3.47 × 10³/mL [small] vs. 2.30 × 10³/mL [large]; p = 0.0288) (Fig. 6C). Regarding endothelial cell precursors, we found more CD31posCD45negCD14negCD133negCD34pos ECFCs (average of 1.41 × 10⁵ [small] vs. 1.05 × 10⁵/mL [large]; p < 0.0001) per milliliter in small volume aspirates compared to large volume aspirates (Additional file 1: Fig. S9).

Next, we asked, if the lower concentration of the nucleated cells that we observed in the large volume aspirates could come with a higher plasma fraction or more RBCs that would compensatory add up to the aspirate volume. Therefore, we analyzed the Hct as well as the Hb and RBC concentrations. We recorded higher Hct values (average of 37.52% [large] vs. 36.40% [small]; p = 0.0370) in the large volume aspirates (Fig. 7A), which excluded a substantial plasma dilution in these samples, but pointed toward RBCs as the main source contributing to the lower TNC concentrations in the large volume aspirates. Indeed, we found more RBCs per milliliter (average of 4.307 × 10⁹ [large] vs. 4.150 × 10⁹ [small]; p = 0.0297) (Fig. 7B) and more Hb per deciliter (average of 13.08 g [large] vs. 12.67 g [small]; p = 0.0392) in the large volume aspirates than in small volume aspirates (Fig. 7C), where the latter did not change during the time course of the BM collection process (Fig. 7D).

Manufacture of cell therapeutics with small volume bone marrow aspirates substantially reduces the required bone marrow volume, increases manufacture efficiency, and minimizes hemoglobin loss for donors.

We observed that small volume aspirates concentrate nucleated cells, including HSPCs and MSCs, and contain less Hb per volume unit. In order to assess the clinical relevance of our findings, we projected the required BM volume and expected Hb loss for various TNC and CD34pos HPC-M target doses for both small and large volume aspirates. We found that small volume BM aspirates save up to 42% BM volume and 44% Hb for HPC-M donors with an increasing saving effect at higher patient’s body weight (Fig. 8). We also tested if small volume aspirates would be favorable for BM-MSC therapeutics production. Projecting MSC doses that could be manufactured from a given BM volume, we found that the production efficiency was more than 150% higher, for both pediatric and adult patients, with small volume aspirates compared to large volume aspirates (Table 2).