Use of a commercial methylcellulose medium with and without recombinant bovine granulocyte colony-stimulating factor for culturing bovine bone marrow cells

Abstract

A commercial methylcellulose culture medium, with and without the addition of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF), was utilized for culturing bovine bone marrow cells in a colony-forming unit assay. Bone marrow mononuclear cells were isolated and cultured in a commercial methylcellulose-based medium containing several recombinant human cytokines. Cultures were prepared with and without 100 ng/mL of rbG-CSF. The size and mean number of colonies per plate from culture days 3 to 9 were compared. We concluded that bovine bone marrow colony growth was supported by this culture medium. The addition of rbG-CSF yielded larger and more numerous colonies. There were significantly more colonies on day 3 (P < 0.001), day 4 (P < 0.001), and day 5 (P = 0.03) with rbG-CSF. Both culture media had the highest colony counts on day 5.

The objective of this study was to utilize a commercial methylcellulose culture medium, with and without the addition of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF), for culturing bovine bone marrow cells in a colony-forming unit (CFU) assay. A CFU assay is a common approach used to study hematopoiesis. It assesses the proliferative capacity of the bone marrow at a given point in time.

Researchers have previously cultured bovine bone marrow cells using a combination of laboratory prepared methylcellulose, fetal bovine serum (FBS), and lymphocyte-conditioned medium (LCM), which contains cytokines harvested from Concanavalin A-stimulated bovine mononuclear cells (1–5). Although LCM and FBS supported colony growth, the variation between LCM preparations and lot numbers of FBS decrease the reproducibility of the assay. Therefore, using a standardized, prepared commercial medium and recombinant cytokines might be preferable. Further advantages include decreased risk of contamination, convenience, and availability of a uniform product for the duration of the research work.

Using a commercial medium for bovine hematopoietic cultures has not been previously described. In addition to methylcellulose, the commercial product (Methocult GF H4534) also contains FBS, bovine serum albumin (BSA), 2-mercaptoethanol, L-glutamine, and 3 recombinant human (rh) cytokines (rh stem cell factor [SCF], rh granulocyte-macrophage colony-stimulating factor [GM-CSF], and rh interleukin-3 [IL-3]). This combination optimizes growth of human granulocyte, macrophage, and granulocyte-macrophage CFU (CFU-G, CFU-M, and CFU-GM, respectively) in culture (6). We wanted to investigate if the medium would support bovine colony growth and whether adding rbG-CSF would improve the growth of CFU-G and CFU-GM colonies. Researchers have studied the actions of rbG-CSF on bovine neutrophils (7,8) and its potential use as an immunomodulator (9,10). However, its potential use in bovine hematopoietic cultures has not been previously described.

All animal procedures were approved by the Canadian Council on Animal Care and followed the Guide to the Care and Use of Experimental Animals (11). Bone marrow (3 mL) was collected from the sternebrae of 6- to 8-month-old Holstein steers that were sedated with 0.25 mg/kg xylazine given intramuscularly (Rompun; Bayer, Toronto, Ontario). The marrow was collected into a 12 mL syringe and immediately transferred into 6 mL of collection medium consisting of minimum essential medium (Iscove’s Modified Dulbecco’s Minimal Essential Medium [IMDM]; Invitrogen-Gibco, Burlington, Ontario) with 584 mg/L of L-glutamine; 10% heat inactivated FBS (CanSera, Rexdale, Ontario); 100 IU penicillin G/0, 1 mg streptomycin sulfate, and 0.25 μg amphotericin B per mL (Antibiotic Antimycotic Solution; Sigma-Aldrich Canada, Oakville, Ontario); and 20 IU/mL of lithium heparin (Heparin Lithium Salt; Sigma-Aldrich). Before collecting the marrow, 1 mL of this collection medium was aspirated into the 12 mL syringe to prevent clotting.

Bone marrow mononuclear cells were isolated by centrifugation of the suspension on 10 mL of Ficoll-Hypaque (Histopaque-1077; Sigma-Aldrich) for 30 min at 800 × g at room temperature (RT). The interface layer was collected and washed twice with an IMDM suspension containing the components described above without heparin. Erythrocytes were lysed by exposure to 10 mL of cold NH₄Cl buffer (0.15 M NH4Cl, 10 mM NaHCO₃) for 5 min. Between each washing and the erythrocyte lysis step, cells were pelleted by centrifugation (400 × g, 10 min, RT). Finally, the cell pellet was resuspended in 1 mL of IMDM and 10% heat inactivated FBS.

Cell viability was assessed by trypan blue exclusion. The total cell count was determined using a hemocytometer. The cells were added to each culture medium in a ratio of 1 part cells to 9 parts medium to give final concentrations of 1 × 10⁵ cells/mL and 0.9% methylcellulose. A culture medium (Methocult GF H4534; StemCell Technologies, Vancouver, British Columbia) was used, with and without the addition of 100 ng/mL of rbG-CSF (Amgen, Thousand Oaks, California, USA), for each bone marrow sample. This concentration of rbG-CSF was chosen arbitrarily based upon preliminary work and a previous study utilizing recombinant human G-CSF for culturing bovine progenitor cells (12). Before plating, the media and cells were vortexed until completely mixed, followed by 10 min settling time to allow for air bubbles to surface. Aliquots of 1 mL of the mixture were plated in 35 × 10 mm petri-dishes with grids (Nalge Nunc International Rochester, New York, USA) to facilitate counting. The cultures were incubated in a humidified environment at 37°C with 5% CO₂ in room air. Colonies of 20 or more cells were counted using an inverted microscope every 24 h from culture day 3 to day 9. On day 9, direct smears were prepared by transferring individual colonies to a glass slide using a 10 μL pipette in order to identify colonies on the basis of cell morphology and peroxidase staining (Myeloperoxidase Kit; Sigma-Aldrich).

Overall, 16 individual bone marrow samples were obtained from 5 calves (n = 5). Bone marrow mononuclear cells from each sample were plated in media, with and without rbG-CSF, in duplicate. Therefore, 64 cultures were prepared, half containing rbG-CSF. Cultures failed to grow on 1 sampling day and 4 culture plates (1 without rbG-CSF, 3 with rbG-CSF) ceased to grow at various stages of the experiment.

Data were analyzed using repeated-measures analysis of variance (ANOVA) with the Poisson regression model (SAS for Windows, version 8.2; SAS Institute, Cary, North Carolina, USA). Then, multiple comparisons using multivariate t-tests were used to compare the differences in the mean number of colonies per plate between the 2 media on each culture day, from days 3 to 9. Differences with P ≤ 0.05 were considered significant.

Although the commercial medium alone supported colony growth, the addition of rbG-CSF made a substantial difference. There were significantly more colonies on day 3 (P < 0.001), day 4 (P < 0.001), and day 5 (P = 0.03) when rbG-CSF was added compared to the commercial medium alone (Figure 1). There were approximately 2 to 8 times more colonies when rbG-CSF was added, with the most dramatic difference noted on day 3 (Table I). There were no significant differences found on days 6, 7, 8, and 9 during the time all colony counts were decreasing. Both culture media had the highest counts on day 5 (Figure 1).

Figure 1.

Mean number of colonies per plate from culture day 3 to 9 in each culture media^a (grey = Methocult GF H4534, black = Methocult GF H4534 with 100 ng/mL rbG-CSF).

Each bar represents the mean of 5 calves (each calf sampled 3 times and each sample plated in duplicate) with 95% confidence interval error bars.

^a All cultures plated with 1 × 105 bovine bone marrow mononuclear cells/mL medium.

* Significant difference (P ≤ 0.05) in colony counts between media for that culture day.

Table I.

Comparison of the ratio of mean number of colonies per plate between 2 culture media^a on culture days 3 to 9

Culture day	Ratio	LLb	ULb	P-value
3	8.28	3.98	17.23	< 0.001c
4	2.71	1.57	4.69	< 0.001c
5	1.74	1.03	2.94	0.03c
6	1.68	0.81	3.50	0.06
7	1.54	0.89	2.67	0.20
8	1.55	0.87	2.78	0.25
9	1.82	0.95	3.49	0.09

^aMethocult GF H4534 with the addition of 100 ng/mL rbG-CSF compared to Methocult GF H4534 alone. All cultures plated in duplicate with 1 × 10⁵ bovine bone marrow mononuclear cells/mL medium

^bLower limit (LL) and upper limit (UL) of the 95% confidence interval

^cP ≤ 0.05 considered significantly different (2-tailed P-value)

Colony size was approximately 2 to 5 times larger with the addition of rbG-CSF (Figure 2). Approximately 20 to 100 cells/colony were observed with the commercial medium alone and 40 to 500 cells/colony with the addition of rbG-CSF (data not shown).

Figure 2.

Bovine bone marrow colonies on culture day 5 using Methocult GF H5434 (A, left) and Methocult GF H5434 with 100 ng/mL rbG-CSF (B, right). Bar = 100 μm.

Although the size and the number of colonies were greater with rbG-CSF, the types of colonies identified were similar. Both CFU-G and CFU-M colonies were observed, but the CFU-GM colonies predominated and were identified based on cell morphology and differences in peroxidase staining. Bovine monocytes have minimal to no peroxidase, while granulocytic cells contain peroxidase, beginning at the promyelocyte stage (13).

This is the first report of a commercial human bone marrow culture medium used for culturing bovine bone marrow cells. We found that the medium (Methocult GF H4534) supported growth of bovine CFU. The colonies obtained were similar to those previously described using laboratory prepared media (1–3). In pilot studies, we also confirmed that similar colonies may be cultured using various in-house prepared methylcellulose media with FBS and LCM, however, colony growth was much less predictable and often unsuccessful. Although the cost of a commercial culture medium may be higher, it is convenient to use and avoids the difficulty of preparing a methylcellulose medium. The addition of rbG-CSF significantly enhanced both the quantity and quality of CFU, making this a useful medium for a bovine CFU-assay.