Biopsy Needle Advancement during Bone Marrow Aspiration Increases Mesenchymal Stem Cell Concentration

Abstract

Point-of-care kits to concentrate bone marrow (BM)-derived mesenchymal stem cells (MSCs) are used clinically in horses. A maximal number of MSCs per milliliter of marrow aspirated might be desired prior to use of a point-of-care system to concentrate MSCs. Our objective was to test a method to increase the number of MSCs per milliliter of marrow collected. We collected two BM aspirates using two different collection techniques from 12 horses. The first collection technique was to aspirate BM from a single site without advancement of the biopsy needle. The second collection technique was to aspirate marrow from multiple sites within the same sternal puncture by advancing the needle 5 mm three times for BM aspiration from four sites. Numbers of MSCs in collected BM were assessed by total nucleated cell count of BM after aspiration, total colony-forming unit-fibroblast (CFU-F) assay, and total MSC number at each culture passage. The BM aspiration technique of four needle advancements during BM aspiration resulted in higher initial nucleated cell counts, more CFU-Fs, and more MSCs at the first passage. There were no differences in the number of MSCs at later passages. Multiple advancements of the BM needle during BM aspiration resulted in increased MSC concentration at the time of BM collection. If a point-of-care kit is used to concentrate MSCs, multiple advancements may result in higher MSC numbers in the BM concentrate after preparation by the point-of-care kit. For culture expanded MSCs beyond the first cell passage, the difference is of questionable clinical relevance.

Keywords: MSC; aspirate; cell-based therapy; horse; point-of-care; regenerative medicine.

Figure 1

Photograph of bone marrow (BM) aspiration needle showing (A) stylet, (B) needle, (C) aspiration holes, and (D) centimeter marks used to measure 2 cm distance from the ventral cortical surface of the sternum.

Figure 2

Colony-forming-unit-fibroblast (CFU-F) assay from bone marrow aspirates using a single site (SS) or a multiple site (MS) technique during bone marrow aspiration (mean, standard deviation). There were significantly more CFU-Fs from bone marrow collected with the MS technique (p = 0.02, paired t-test).

Figure 3

Total nucleated cell count (mean, standard deviation) per milliliter of raw bone marrow aspirated using a single site (SS) or a multiple site (MS) technique during bone marrow aspiration. There were significantly more nucleated cells in BM collected with the MS technique (p = 0.01, paired t-test).

Figure 4

Total number of mesenchymal stem cells (MSCs; mean, standard deviation) at the each passage from bone marrow aspirates using a single site (SS) or a multiple site (MS) technique during bone marrow (BM) aspiration. There were significantly more MSCs from BM collected with the MS technique (p = 0.02, Wilcoxon-signed rank test). The number of MSCs at passages 2 and 3 were not significantly different between SS and MS techniques (p = 0.1; p = 0.2).

Figure 5

Examples of MSCs after (A) adipogenic, (B) osteogenic, and (C) chondrogenic induction and staining with resulting in lipid, calcium, and proteoglycan accumulation. Original magnification 200× (A,C) and 40× (B); scale bar = 100 μm (A,C) or 500 μm (B).