JIM-11859; No of Pages 5 Journal of Immunological Methods xxx (2014) xxx–xxx
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Technical note
A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques Mats Spångberg a,⁎, Paola Martinez b, Helene Fredlund a, Gunilla B. Karlsson Hedestam b, Christopher Sundling b,⁎⁎ a b
Comparative Medicine, Astrid Fagraeus Laboratory, Karolinska Institutet, Stockholm, Sweden Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
a r t i c l e
i n f o
Article history: Received 11 March 2014 Received in revised form 9 May 2014 Accepted 9 May 2014 Available online xxxx Keywords: Bone marrow aspirate Technique Non-human primate Macaque B cell Plasma cell
a b s t r a c t Longitudinal bone marrow aspirates were obtained aseptically from the humerus of 36 rhesus and 6 cynomolgus macaques by using a 20G spinal needle, introduced through the bone close to the greater tuberosity. All samplings were performed without complications, and the animals showed no signs of pain or infections. The amount of total bone marrow cells obtained from each aspiration varied, in part due to animal-to-animal variation, but the yields were not affected by the sampling frequency or the length of time between each aspiration. The frequency of plasma cells in the bone marrow of each animal was also fairly stable over several longitudinal samplings while a greater, age-dependent, variation was observed between different animals. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The non-human primate (NHP) model is used for diverse applications in experimental medicine. The NHP species that are most commonly used include rhesus and cynomolgus macaques, which possess high genetic homology to humans (Gibbs et al., 2007). In addition to peripheral blood cells, bone marrow aspirates are readily collected from NHPs as described in studies of mesenchymal and hematopoietic stem cells (Shields et al., 2005; Sharma et al., 2011), investigations of host responses to vaccination (Sundling et al., 2010, 2013), and reports of the
Abbreviations: NHP, Non-human primate; ASC, Antibody-secreting cell. ⁎ Correspondence to: M. Spångberg, Comparative Medicine, Astrid Fagraeus Laboratory, Karolinska Institutet, 171 77 Stockholm, Sweden. Tel.: +46 8 524 85 973. ⁎⁎ Correspondence to: C. Sundling, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 77 Stockholm, Sweden. Tel.: +46 8 524 86 943. E-mail addresses: [email protected] (M. Spångberg), [email protected] (C. Sundling).
recovery of hematopoietic cells for subsequent transplantation (Ueda et al., 2004). The procedures used for propagating bone marrow cells are well described in the literature, but the sampling techniques used to retrieve the bone marrow are often insufficiently described, and in some cases associated with side effects (Llanos et al., 2006). Here, we describe a protocol for collecting longitudinal bone marrow aspirates from the humerus of rhesus and cynomolgus macaques, which in our hands is easy to perform and inflicts minimal harm to the animals. We further describe the yields of bone marrow cells and antibody-secreting plasma cells that can be obtained from longitudinal sampling and we discuss possible sources of variation in cell numbers obtained from this method of sampling. 2. Methods 2.1. Animals and sampling Bone marrow aspirates were obtained from the humerus of 36 rhesus and 6 cynomolgus macaques derived from three
http://dx.doi.org/10.1016/j.jim.2014.05.004 0022-1759/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Spångberg, M., et al., A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.05.004
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M. Spångberg et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
separate studies, NHP09 (n = 12 rhesus macaques) (Sundling et al., 2010), NHP10 (n = 6 cynomolgus macaques) (Sundling et al., 2013), and NHP11 (n = 24 rhesus macaques) (Martinez et al., unpublished data). Each animal was sampled 2–7 times over the course of 8–37 weeks depending on the design of the study. All macaques were females between three to ten years old. All procedures were approved by the Local Ethical Committee on Animal Experiments in Stockholm, Sweden. 2.2. Bone marrow aspiration Before sampling, the animals were anaesthetized by intramuscular injection of 10–15 mg/kg of ketamin and 0.5 mg/kg of xylazine. Carprofen was given subcutaneously as analgesia at a dose of 3–4 mg/kg. A strict aseptic technique was used throughout the procedure. Syringes and needles used for the sampling were flushed with a heparin solution. A 20G × 1 1/2″ spinal needle with an attached stylet was inserted through the skin and into the bone 2–3 mm medially to the greater tuberosity of the humerus. By imparting torque, the needle was introduced approximately 15 mm into the humerus until it was firmly engaged in the bone. A 5 ml syringe was attached to the needle, and 1 to 1.5 ml of aspirate was sampled by applying a vacuum in the syringe. With the needle still in place, the syringe was removed and the aspirate emptied into a 9 ml EDTA-coated plastic tube. The procedure was then repeated until a total of 5– 6 ml was sampled. Bone marrow aspirates were sampled from alternating arms of the macaques at a maximum interval of 4 weeks (in the NHP10 group; see Section 2.1), giving an 8 weeks period of recovery after sampling. 2.3. Purification of mononuclear cells The mononuclear cells were purified from the bone marrow aspirates through density gradient centrifugation with Ficoll Paque Plus (GE Healthcare), after which red blood cells were removed by lysis with NH4Cl followed by extensive washing with PBS. The number of cells was counted and the viability was assessed by labeling with Trypan Blue. Cells were then frozen in fetal bovine serum supplemented with 10% DMSO. 2.4. B cell ELISpot To detect IgG-producing cells in the bone marrow aspirates, ELISpot plates were coated with 10 μg/ml anti-human Fcγ (Jackson Immunoresearch) overnight at 4 °C. Following washing of the plates in PBS supplemented with 0.05% Tween-20, cells were plated in complete media in a 5-fold dilution series starting at 500,000 cells in the top row and incubated overnight at 37 °C, 5% CO2. Fresh cells were plated directly, while frozen cells were first thawed in a 37 °C water bath, washed three times in complete media, and left resting over the day before plating. The next day, following washing, the plates were incubated with 0.25 μg/ml biotinylated goat-antihuman Fcγ antibody (Jackson Immunoresearch) for 1.5 h before washing and addition of Streptavidin–Alkaline phosphatase (Mabtech) diluted 1/1000 in PBS and incubation for 45 min. Plates were then washed and spots were visualized by addition of BCIP/NBT substrate (Sigma) for 5 min before stopping the reaction by extensive washing with water. After drying, spots corresponding to antibody-secreting cells (ASCs)
were counted using an Immunospot analyzer (Cellular Technology Ltd.) and the amount of ASC per 106 plated bone marrow cells was calculated. 2.5. Statistics All statistical evaluations were done using non-parametric tests. In comparisons including three or more groups or time points, the unpaired Kruskal–Wallis or paired Friedman tests were used followed by Dunn's test for multiple comparisons. When two groups were compared, the Mann–Whitney test was used. Correlations were determined with linear regression and calculation of Spearman r. All tests were done using GraphPad Prism version 6 software. 3. Results 3.1. Purified mononuclear cell yields from bone marrow aspirates A total of 142 bone marrow aspirates were obtained from the humerus of 36 rhesus macaques (100 aspirates) and 6 cynomolgus macaques (42 aspirates). All samplings were performed without complications and the animals showed no signs of pain or infections. The number of mononuclear cells in the aspirates varied between 11 × 106 and 570 × 106 per sample, with a median yield of 55 × 106 (Fig. 1A). No difference in yield was observed between the rhesus and cynomolgus groups. The viability of the cells was generally more than 95%. Memory B cells were previously shown to be undetectable in the bone marrow aspirates, indicating that no contamination with peripheral blood occurs during sampling (Sundling et al., 2010). 3.2. Effect of serial sampling on bone marrow mononuclear cell yields To determine if serial sampling of the bone marrow would lead to reduced yields of isolated cells, the number of purified mononuclear cells obtained at each sampling was pooled and statistically evaluated using the Kruskal–Wallis test followed by Dunn's test for multiple comparisons (Fig. 1B). No difference was observed between the samplings, indicating that repeated sampling had no detrimental effects on the bone marrow cell yields. To further investigate if the time between each sampling would affect the yield, the macaques were separated into groups based on sampling frequency and interval (Fig. 1C). The 12 rhesus macaques in NHP09 (blue triangles) were sampled 2 times separated by a 4-week interval, the 6 cynomolgus macaques in NHP10 (red circles) were sampled 7 times separated by 4–5 week intervals, and the 18 rhesus macaques in NHP11 (black boxes) were sampled 4 times separated by 4–20 week intervals. Each group was evaluated with the matched-pair Friedman test followed by Dunn's test for multiple comparisons. A significant reduction in the yield of bone marrow cells was observed following the second sampling in the NHP10 group (Fig. 1C), but this likely reflected a temporary drop as the continued sampling time points resulted in higher cell numbers.
Please cite this article as: Spångberg, M., et al., A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.05.004
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Fig. 1. Isolation of mononuclear cells from bone marrow aspirations. (A) The individual numbers (dots) and median yield (line) of purified mononuclear cells from rhesus (n = 100) and cynomolgus (n = 42) macaques are shown. (B) Each macaque was sampled 2–7 times and the number of isolated cells at each time-point is shown. The individual samples (open circles) and geometric mean (red line) are indicated. (C) Aspirates were separated into three groups; NHP09 (blue triangles, n = 12), NHP10 (red circles, n = 6), and NHP11 (black boxes, n = 18) based on sampling frequency and interval, where weeks since the first bone marrow sampling is indicated by the X-axis. Differences were evaluated by the matched-pair non-parametric Friedman test, followed by Dunn's test for multiple comparisons, where *p b 0.05 and **p b 0.01.
3.3. Enumeration of plasma cells in the bone marrow aspirates We next enumerated plasma cell numbers in the mononuclear cells isolated from the bone marrow aspirates by B cell ELISpot assay. A total of 90 samples were analyzed from 28 macaques (22 rhesus and 6 cynomolgus) with a median of 2365 antibody-secreting cells (ASCs) detected per million plated bone marrow cells, corresponding to a frequency of 0.24% (Fig. 2A), which is in line with previous reports on bone marrow resident plasma cell frequencies in humans (Terstappen et al., 1990). There was considerable variation in the numbers of ASC, ranging between 245 and 42,496 ASC per million plated mononuclear cells (coefficient of variation of 146%), which was similar to the variation observed in the total bone marrow counts (coefficient of variation of 98.4%). The two compartments were only weakly correlated, however (Spearman r 0.28, p b 0.0130, data not shown). In contrast, the plasma cell numbers correlated strongly with the age of the animals (Fig. 2B; Spearman r 0.69, p b 0.0001), although the spread in age was mainly divided into the three groups; NHP09 (blue; mean age 2.8 years), NHP10 (red; mean age 9.3 years) and NHP11 (black; mean age 5.7 years), with limited variation within the groups. 3.4. Effect of serial sampling on bone marrow plasma cell numbers To determine if the sample interval and frequency affected the bone marrow plasma cell numbers, the animals were
grouped according to sample frequency and interval as in Fig. 1C, with weeks following the first bone marrow aspiration shown on the X-axis. NHP10 (red) were sampled four times, NHP11 (black) three times, and NHP09 (blue) two times (Fig. 2C). Similar to the total bone marrow counts there was a drop in the measured plasma cell numbers at the second sampling point in the NHP10 group, likely representing suboptimal handling of the cells at this time point. Overall, there was no effect of sampling interval or frequency on plasma cell numbers. To investigate the animal-specific contribution, the plasma cell yields from different time points for individual animals were compared (Fig. 2D). Although there was a considerable variation in plasma cell numbers in the data set as a whole (Fig. 2A), there was less variation between different sampling time points for each individual animal (Fig. 2C, D), supporting an animal-specific contribution to the plasma cell numbers measured. 4. Discussion The bone marrow is a highly specialized immune compartment and an important niche for hematopoietic stem cells and long-lived plasma cells. For vaccines aimed at stimulating protective B cell responses, assessment of elicited immune responses is often limited to serological measurements of antibody titers and, in some cases, enumeration of peripheral memory B cells in circulating blood. However, for a more
Please cite this article as: Spångberg, M., et al., A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.05.004
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the number of plasma cells in 90 bone marrow aspirates. Although there was considerable variation in the number of cells obtained, partly due to animal-specific variation, there was no obvious detrimental effect of repeated sampling, even when performed every 4 weeks. This is consistent with previous studies detailing repeated evaluation of antigen-specific plasma cells in immunized macaques (Sundling et al., 2010, 2013). Interestingly, we found that the plasma cell numbers correlated with the age of the macaques at the time of sampling, potentially indicating that new plasma cell reactivities are gained as the animals are exposed to new infectious agents over time. These results establish this technique as a robust approach for longitudinal assessment of bone marrow-resident plasma cells in NHPs and allow for a more complete understanding of this specialized immune compartment and of long-lived antibody responses. Acknowledgments We appreciate the help and technical expertise generously provided by Martje Fentener van Vlissingen, Dominique Kuiper and Dennis de Meulder at Erasmus Lab Animal Science Center, Rotterdam, The Netherlands. Fig. 2. Plasma cell enumeration in bone marrow aspirates. (A) IgG-secreting plasma cells were enumerated by B cell ELISpot assay for 90 bone marrow aspirates derived from 28 macaques, with the median indicated by the line. (B) Correlation between the plasma cell numbers and age at the time of sampling was determined with linear regression and calculation of the non-parametric Spearman r. The samples are colored to indicate NHP09 (blue), NHP10 (red), and NHP11 (black) origin. (C) Longitudinal plasma cell measurements are shown for the individual macaques. The animals are colored red (NHP10), black (NHP11) or blue (NHP09) depending on the number of samplings, with 4, 3, and 2 measurements respectively. The X-axis indicates weeks after the first bone marrow aspiration. (D) To evaluate the variability of plasma cell numbers in individual donors, all measurements performed for each animal (open circles) were pooled and presented as bar graphs, color-coded as in C, indicating the mean + SEM.
complete understanding of the capacity of a given vaccine candidate to elicit long-lived B cell responses, measurements of long-lived antigen-specific plasma cells would add significant value. Methods to analyze cells in the bone marrow, where fully differentiated long-lived plasma cells reside (Tarlinton et al., 2008), are therefore needed. Long-lived plasma cells play an important role in the protection against re-infection through their capacity to secrete antibodies that provide first line serological defenses. Long-lived, bone marrow-resident plasma cells were first described in mice (Slifka et al., 1998), and are also easily identified in human bone marrow (Rawstron, 2006), but they are less well characterized in NHPs. The availability of a simple and reliable technique for collecting bone marrow aspirates from rhesus or cynomolgus macaques is therefore highly valuable for basic studies of bone marrow cells in experimental settings. The technique described here allows for longitudinal bone marrow sampling yielding high numbers of cells for subsequent freezing or for direct functional analysis in vitro. Using this technique we present data from 142 bone marrow aspirations from 36 macaques, each sampled 2–7 times with different intervals between the samplings. We further determined
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Ueda, K., Hanazono, Y., Shibata, H., Ageyama, N., Ueda, Y., Ogata, S., Tabata, T., Nagashima, T., Takatoku, M., Kume, A., Ikehara, S., Taniwaki, M., Terao, K., Hasegawa, M., Ozawa, K., 2004. High-level in vivo gene marking after gene-modified autologous hematopoietic stem cell transplantation without marrow conditioning in nonhuman primates. Mol. Ther. 10, 469.
Please cite this article as: Spångberg, M., et al., A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.05.004
Contents lists available at ScienceDirect
Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim
Technical note
A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques Mats Spångberg a,⁎, Paola Martinez b, Helene Fredlund a, Gunilla B. Karlsson Hedestam b, Christopher Sundling b,⁎⁎ a b
Comparative Medicine, Astrid Fagraeus Laboratory, Karolinska Institutet, Stockholm, Sweden Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
a r t i c l e
i n f o
Article history: Received 11 March 2014 Received in revised form 9 May 2014 Accepted 9 May 2014 Available online xxxx Keywords: Bone marrow aspirate Technique Non-human primate Macaque B cell Plasma cell
a b s t r a c t Longitudinal bone marrow aspirates were obtained aseptically from the humerus of 36 rhesus and 6 cynomolgus macaques by using a 20G spinal needle, introduced through the bone close to the greater tuberosity. All samplings were performed without complications, and the animals showed no signs of pain or infections. The amount of total bone marrow cells obtained from each aspiration varied, in part due to animal-to-animal variation, but the yields were not affected by the sampling frequency or the length of time between each aspiration. The frequency of plasma cells in the bone marrow of each animal was also fairly stable over several longitudinal samplings while a greater, age-dependent, variation was observed between different animals. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The non-human primate (NHP) model is used for diverse applications in experimental medicine. The NHP species that are most commonly used include rhesus and cynomolgus macaques, which possess high genetic homology to humans (Gibbs et al., 2007). In addition to peripheral blood cells, bone marrow aspirates are readily collected from NHPs as described in studies of mesenchymal and hematopoietic stem cells (Shields et al., 2005; Sharma et al., 2011), investigations of host responses to vaccination (Sundling et al., 2010, 2013), and reports of the
Abbreviations: NHP, Non-human primate; ASC, Antibody-secreting cell. ⁎ Correspondence to: M. Spångberg, Comparative Medicine, Astrid Fagraeus Laboratory, Karolinska Institutet, 171 77 Stockholm, Sweden. Tel.: +46 8 524 85 973. ⁎⁎ Correspondence to: C. Sundling, Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, 171 77 Stockholm, Sweden. Tel.: +46 8 524 86 943. E-mail addresses: [email protected] (M. Spångberg), [email protected] (C. Sundling).
recovery of hematopoietic cells for subsequent transplantation (Ueda et al., 2004). The procedures used for propagating bone marrow cells are well described in the literature, but the sampling techniques used to retrieve the bone marrow are often insufficiently described, and in some cases associated with side effects (Llanos et al., 2006). Here, we describe a protocol for collecting longitudinal bone marrow aspirates from the humerus of rhesus and cynomolgus macaques, which in our hands is easy to perform and inflicts minimal harm to the animals. We further describe the yields of bone marrow cells and antibody-secreting plasma cells that can be obtained from longitudinal sampling and we discuss possible sources of variation in cell numbers obtained from this method of sampling. 2. Methods 2.1. Animals and sampling Bone marrow aspirates were obtained from the humerus of 36 rhesus and 6 cynomolgus macaques derived from three
http://dx.doi.org/10.1016/j.jim.2014.05.004 0022-1759/© 2014 Elsevier B.V. All rights reserved.
Please cite this article as: Spångberg, M., et al., A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.05.004
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M. Spångberg et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
separate studies, NHP09 (n = 12 rhesus macaques) (Sundling et al., 2010), NHP10 (n = 6 cynomolgus macaques) (Sundling et al., 2013), and NHP11 (n = 24 rhesus macaques) (Martinez et al., unpublished data). Each animal was sampled 2–7 times over the course of 8–37 weeks depending on the design of the study. All macaques were females between three to ten years old. All procedures were approved by the Local Ethical Committee on Animal Experiments in Stockholm, Sweden. 2.2. Bone marrow aspiration Before sampling, the animals were anaesthetized by intramuscular injection of 10–15 mg/kg of ketamin and 0.5 mg/kg of xylazine. Carprofen was given subcutaneously as analgesia at a dose of 3–4 mg/kg. A strict aseptic technique was used throughout the procedure. Syringes and needles used for the sampling were flushed with a heparin solution. A 20G × 1 1/2″ spinal needle with an attached stylet was inserted through the skin and into the bone 2–3 mm medially to the greater tuberosity of the humerus. By imparting torque, the needle was introduced approximately 15 mm into the humerus until it was firmly engaged in the bone. A 5 ml syringe was attached to the needle, and 1 to 1.5 ml of aspirate was sampled by applying a vacuum in the syringe. With the needle still in place, the syringe was removed and the aspirate emptied into a 9 ml EDTA-coated plastic tube. The procedure was then repeated until a total of 5– 6 ml was sampled. Bone marrow aspirates were sampled from alternating arms of the macaques at a maximum interval of 4 weeks (in the NHP10 group; see Section 2.1), giving an 8 weeks period of recovery after sampling. 2.3. Purification of mononuclear cells The mononuclear cells were purified from the bone marrow aspirates through density gradient centrifugation with Ficoll Paque Plus (GE Healthcare), after which red blood cells were removed by lysis with NH4Cl followed by extensive washing with PBS. The number of cells was counted and the viability was assessed by labeling with Trypan Blue. Cells were then frozen in fetal bovine serum supplemented with 10% DMSO. 2.4. B cell ELISpot To detect IgG-producing cells in the bone marrow aspirates, ELISpot plates were coated with 10 μg/ml anti-human Fcγ (Jackson Immunoresearch) overnight at 4 °C. Following washing of the plates in PBS supplemented with 0.05% Tween-20, cells were plated in complete media in a 5-fold dilution series starting at 500,000 cells in the top row and incubated overnight at 37 °C, 5% CO2. Fresh cells were plated directly, while frozen cells were first thawed in a 37 °C water bath, washed three times in complete media, and left resting over the day before plating. The next day, following washing, the plates were incubated with 0.25 μg/ml biotinylated goat-antihuman Fcγ antibody (Jackson Immunoresearch) for 1.5 h before washing and addition of Streptavidin–Alkaline phosphatase (Mabtech) diluted 1/1000 in PBS and incubation for 45 min. Plates were then washed and spots were visualized by addition of BCIP/NBT substrate (Sigma) for 5 min before stopping the reaction by extensive washing with water. After drying, spots corresponding to antibody-secreting cells (ASCs)
were counted using an Immunospot analyzer (Cellular Technology Ltd.) and the amount of ASC per 106 plated bone marrow cells was calculated. 2.5. Statistics All statistical evaluations were done using non-parametric tests. In comparisons including three or more groups or time points, the unpaired Kruskal–Wallis or paired Friedman tests were used followed by Dunn's test for multiple comparisons. When two groups were compared, the Mann–Whitney test was used. Correlations were determined with linear regression and calculation of Spearman r. All tests were done using GraphPad Prism version 6 software. 3. Results 3.1. Purified mononuclear cell yields from bone marrow aspirates A total of 142 bone marrow aspirates were obtained from the humerus of 36 rhesus macaques (100 aspirates) and 6 cynomolgus macaques (42 aspirates). All samplings were performed without complications and the animals showed no signs of pain or infections. The number of mononuclear cells in the aspirates varied between 11 × 106 and 570 × 106 per sample, with a median yield of 55 × 106 (Fig. 1A). No difference in yield was observed between the rhesus and cynomolgus groups. The viability of the cells was generally more than 95%. Memory B cells were previously shown to be undetectable in the bone marrow aspirates, indicating that no contamination with peripheral blood occurs during sampling (Sundling et al., 2010). 3.2. Effect of serial sampling on bone marrow mononuclear cell yields To determine if serial sampling of the bone marrow would lead to reduced yields of isolated cells, the number of purified mononuclear cells obtained at each sampling was pooled and statistically evaluated using the Kruskal–Wallis test followed by Dunn's test for multiple comparisons (Fig. 1B). No difference was observed between the samplings, indicating that repeated sampling had no detrimental effects on the bone marrow cell yields. To further investigate if the time between each sampling would affect the yield, the macaques were separated into groups based on sampling frequency and interval (Fig. 1C). The 12 rhesus macaques in NHP09 (blue triangles) were sampled 2 times separated by a 4-week interval, the 6 cynomolgus macaques in NHP10 (red circles) were sampled 7 times separated by 4–5 week intervals, and the 18 rhesus macaques in NHP11 (black boxes) were sampled 4 times separated by 4–20 week intervals. Each group was evaluated with the matched-pair Friedman test followed by Dunn's test for multiple comparisons. A significant reduction in the yield of bone marrow cells was observed following the second sampling in the NHP10 group (Fig. 1C), but this likely reflected a temporary drop as the continued sampling time points resulted in higher cell numbers.
Please cite this article as: Spångberg, M., et al., A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.05.004
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Fig. 1. Isolation of mononuclear cells from bone marrow aspirations. (A) The individual numbers (dots) and median yield (line) of purified mononuclear cells from rhesus (n = 100) and cynomolgus (n = 42) macaques are shown. (B) Each macaque was sampled 2–7 times and the number of isolated cells at each time-point is shown. The individual samples (open circles) and geometric mean (red line) are indicated. (C) Aspirates were separated into three groups; NHP09 (blue triangles, n = 12), NHP10 (red circles, n = 6), and NHP11 (black boxes, n = 18) based on sampling frequency and interval, where weeks since the first bone marrow sampling is indicated by the X-axis. Differences were evaluated by the matched-pair non-parametric Friedman test, followed by Dunn's test for multiple comparisons, where *p b 0.05 and **p b 0.01.
3.3. Enumeration of plasma cells in the bone marrow aspirates We next enumerated plasma cell numbers in the mononuclear cells isolated from the bone marrow aspirates by B cell ELISpot assay. A total of 90 samples were analyzed from 28 macaques (22 rhesus and 6 cynomolgus) with a median of 2365 antibody-secreting cells (ASCs) detected per million plated bone marrow cells, corresponding to a frequency of 0.24% (Fig. 2A), which is in line with previous reports on bone marrow resident plasma cell frequencies in humans (Terstappen et al., 1990). There was considerable variation in the numbers of ASC, ranging between 245 and 42,496 ASC per million plated mononuclear cells (coefficient of variation of 146%), which was similar to the variation observed in the total bone marrow counts (coefficient of variation of 98.4%). The two compartments were only weakly correlated, however (Spearman r 0.28, p b 0.0130, data not shown). In contrast, the plasma cell numbers correlated strongly with the age of the animals (Fig. 2B; Spearman r 0.69, p b 0.0001), although the spread in age was mainly divided into the three groups; NHP09 (blue; mean age 2.8 years), NHP10 (red; mean age 9.3 years) and NHP11 (black; mean age 5.7 years), with limited variation within the groups. 3.4. Effect of serial sampling on bone marrow plasma cell numbers To determine if the sample interval and frequency affected the bone marrow plasma cell numbers, the animals were
grouped according to sample frequency and interval as in Fig. 1C, with weeks following the first bone marrow aspiration shown on the X-axis. NHP10 (red) were sampled four times, NHP11 (black) three times, and NHP09 (blue) two times (Fig. 2C). Similar to the total bone marrow counts there was a drop in the measured plasma cell numbers at the second sampling point in the NHP10 group, likely representing suboptimal handling of the cells at this time point. Overall, there was no effect of sampling interval or frequency on plasma cell numbers. To investigate the animal-specific contribution, the plasma cell yields from different time points for individual animals were compared (Fig. 2D). Although there was a considerable variation in plasma cell numbers in the data set as a whole (Fig. 2A), there was less variation between different sampling time points for each individual animal (Fig. 2C, D), supporting an animal-specific contribution to the plasma cell numbers measured. 4. Discussion The bone marrow is a highly specialized immune compartment and an important niche for hematopoietic stem cells and long-lived plasma cells. For vaccines aimed at stimulating protective B cell responses, assessment of elicited immune responses is often limited to serological measurements of antibody titers and, in some cases, enumeration of peripheral memory B cells in circulating blood. However, for a more
Please cite this article as: Spångberg, M., et al., A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.05.004
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M. Spångberg et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
the number of plasma cells in 90 bone marrow aspirates. Although there was considerable variation in the number of cells obtained, partly due to animal-specific variation, there was no obvious detrimental effect of repeated sampling, even when performed every 4 weeks. This is consistent with previous studies detailing repeated evaluation of antigen-specific plasma cells in immunized macaques (Sundling et al., 2010, 2013). Interestingly, we found that the plasma cell numbers correlated with the age of the macaques at the time of sampling, potentially indicating that new plasma cell reactivities are gained as the animals are exposed to new infectious agents over time. These results establish this technique as a robust approach for longitudinal assessment of bone marrow-resident plasma cells in NHPs and allow for a more complete understanding of this specialized immune compartment and of long-lived antibody responses. Acknowledgments We appreciate the help and technical expertise generously provided by Martje Fentener van Vlissingen, Dominique Kuiper and Dennis de Meulder at Erasmus Lab Animal Science Center, Rotterdam, The Netherlands. Fig. 2. Plasma cell enumeration in bone marrow aspirates. (A) IgG-secreting plasma cells were enumerated by B cell ELISpot assay for 90 bone marrow aspirates derived from 28 macaques, with the median indicated by the line. (B) Correlation between the plasma cell numbers and age at the time of sampling was determined with linear regression and calculation of the non-parametric Spearman r. The samples are colored to indicate NHP09 (blue), NHP10 (red), and NHP11 (black) origin. (C) Longitudinal plasma cell measurements are shown for the individual macaques. The animals are colored red (NHP10), black (NHP11) or blue (NHP09) depending on the number of samplings, with 4, 3, and 2 measurements respectively. The X-axis indicates weeks after the first bone marrow aspiration. (D) To evaluate the variability of plasma cell numbers in individual donors, all measurements performed for each animal (open circles) were pooled and presented as bar graphs, color-coded as in C, indicating the mean + SEM.
complete understanding of the capacity of a given vaccine candidate to elicit long-lived B cell responses, measurements of long-lived antigen-specific plasma cells would add significant value. Methods to analyze cells in the bone marrow, where fully differentiated long-lived plasma cells reside (Tarlinton et al., 2008), are therefore needed. Long-lived plasma cells play an important role in the protection against re-infection through their capacity to secrete antibodies that provide first line serological defenses. Long-lived, bone marrow-resident plasma cells were first described in mice (Slifka et al., 1998), and are also easily identified in human bone marrow (Rawstron, 2006), but they are less well characterized in NHPs. The availability of a simple and reliable technique for collecting bone marrow aspirates from rhesus or cynomolgus macaques is therefore highly valuable for basic studies of bone marrow cells in experimental settings. The technique described here allows for longitudinal bone marrow sampling yielding high numbers of cells for subsequent freezing or for direct functional analysis in vitro. Using this technique we present data from 142 bone marrow aspirations from 36 macaques, each sampled 2–7 times with different intervals between the samplings. We further determined
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Please cite this article as: Spångberg, M., et al., A simple and safe technique for longitudinal bone marrow aspiration in cynomolgus and rhesus macaques, J. Immunol. Methods (2014), http://dx.doi.org/10.1016/j.jim.2014.05.004
