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Keywords:

  • Bone marrow transplantation;
  • Cynomolgus;
  • Bone marrow harvesting

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

To minimize contamination of bone marrow cells (BMCs) with T cells from the peripheral blood, a new “perfusion method” for collecting BMCs is proposed using cynomolgus monkeys. Two BM puncture needles are inserted into a long bone such as the humerus, femur, or tibia. One needle is connected to an extension tube and the end of the tube is inserted into a culture flask to collect the BM fluid. The other needle is connected to a syringe containing 30 ml of phosphate-buffered saline. The solution is pushed gently from the syringe into the medullary cavity, and the medium containing the BM fluid is collected into the culture flask. There is significantly less contamination with peripheral blood, determined from the frequencies of CD4+ and CD8+ T cells, when using this method (<6%) than when using the conventional method (>20%) consisting of multiple BM aspirations from the iliac crest. Furthermore, the number and progenitor activities of the cells harvested using this “perfusion method” are greater than those harvested using the conventional aspiration method. This perfusion method was carried out 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. These findings suggest that the “perfusion method” is safe and simple and would be of great advantage in obtaining pure BMCs, resulting in a less frequent occurrence of acute graft-versus-host-disease in allogeneic BM transplantation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bone marrow transplantation (BMT) is one of the most powerful strategies for treating leukemia, aplastic anemia, congenital immunodeficiency, and autoimmune diseases [1-3]. Furthermore, gene therapy and organ transplantation using bone marrow cells (BMCs) have recently been carried out [4-8]. In humans, BMCs have usually been collected by multiple BM aspirations from the iliac crest according to the method established by Thomas et al. [9]. However, the peripheral blood has inevitably become a contaminant when using this method, and, because the percentage of T cells in the BMCs is greater than 20%, the result can be acute graft-versus-host disease (GVHD). Therefore, we attempted to establish a new method for collecting BMCs. The method, termed the “perfusion method,” is simple, useful, and minimizes the contamination of the BMCs with T cells [10]. In the present study, using cynomolgus monkeys as a human model, we compare this new method with the conventional aspiration method, and show that the former has great advantages over the latter.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Animals

Normal two- to three-year-old cynomolgus monkeys (2 to 3 kg) were obtained from KEARI (Osaka, Japan). The monkeys were free of intestinal parasites and seronegative for tuberculosis, herpes B, hepatitis A, and hepatitis B virus. All surgical procedures and postoperative care of animals were carried out in accordance with the guidelines of the National Institutes of Health for care and use of primates. The study protocol was approved by the Animal Experimentation Committee, Kansai Medical University.

BMC Harvesting by Perfusion Method

Cynomolgus monkeys were anesthetized using Ketalar® (5 mg, Sankyo Co., Ltd.; Tokyo, Japan; http://www.sankyo.co.jp/menu_e.html), and BM fluid was collected using the following procedure. As shown in Figure 1, two needles (Katsunuma's BM puncture needle [1.8 mm diameter], Kyoto, Japan) were inserted into a long bone such as the humerus, femur, or tibia to the shaft. The end of the extension tube (50 cm, 3.8 ml, Code No. SF-ET3825, Terumo Co., Ltd.; Tokyo, Japan) was connected to a needle. The other end was placed into a culture flask (250 ml, Becton Dickinson; Frankin Lakes, NJ; http://www.bd.com) containing 20 ml of phosphate-buffered saline (PBS) with heparin (10 U/ml, Novo Heparin 1000, Hoechst Marion Roussel Co., Ltd., Tokyo, Japan). The other needle was connected to a syringe (30 ml, Code No. SS-30ES, Terumo Co., Ltd.) containing 30 ml of PBS, and the PBS was then pushed gently from the syringe into the medullary cavity to flush out the BM. The medium containing the BM fluid was collected in the culture flask, and the process was repeated twice.

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Figure Figure 1.. Perfusion method.Two needles were inserted into a humerus (A, B) or a femur (C, D). One needle was connected to an extension tube and the end of the tube was inserted into a culture flask to collect the BM fluid. The other needle was connected to a syringe containing 30 ml of PBS. The solution was pushed gently from the syringe into the BM cavity. The medium containing BM fluid was collected into the flask.

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BMC Harvesting by Conventional Multiple Aspiration Method

BM fluid (total 40 ml) was aspirated from the iliac crest, as previously described [8].

Preparation of BMCs

BMCs, harvested either by the perfusion or aspiration method, were centrifuged and suspended in 15 ml of PBS. They were placed on 15 ml of Lymphoprep density solution (1.077 g/ml, Nycomed Pharma AS; Oslo, Norway). After centrifugation for 30 min at 2,000 rpm at room temperature, the BMCs were collected from the defined layer at the interface.

Isolation of Peripheral Blood Mononuclear Cells (PBMNCs)

PBMNCs were isolated from heparinized blood by centrifugation on a cushion of Lymphoprep density solution.

Analyses of Cell Surface Antigens

Cell surface antigens on the PBMNCs and BMCs were determined using fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-coupled monoclonal antibodies (mAbs) against human CD4, CD8, CD20, CD11b, or CD56 (Exalpha; Boston, MA; http://www.exalpha.com), and IgM (Biosource; Camarillo, CA; http://www.biosource.com/). These mAbs were previously examined for their cross-reactivities to the molecules expressed on cells of cynomolgus monkeys. Flow cytometric analyses were performed using an EPICS-XL® (Coulter Co.; Miami, Florida; http://www.coulter.com).

Colony-Forming Assay

The colony-forming ability of the BMCs (colony-forming units-culture [CFU-C]) was assayed as described previously [11]. Briefly, BMCs (104 cells/wells) were plated in 12-well plates (ICN Biomedicals, Inc.; Aurora, OH; http://www.icnbiomed.com) in a volume of 10 ml of Methocult GF H4434 (Stem Cell Technologies Inc.; Vancouver, BC, Canada; http://www.stemcell.com), consisting of optimal concentrations of cytokines (recombinant human stem cell factor [SCF], erythropoietin [Epo], interleukin 3 [IL-3], GM-CSF, and G-CSF), 30% fetal bovine serum, 1% bovine serum albumin, 2 mML-glutamine, 10–4 M-mercaptoethanol, and 0.9% methylcellulose. Fourteen days later, the CFU-C were counted.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Number of BMCs

BMCs were collected from the long bones using the perfusion method as represented in Figure 1. The number of BMCs from one humerus, femur, or tibia was 4.3 ± 1.4 × 108, 2.7 ± 0.6 × 108, or 0.2 ± 0.1 × 108 cells, respectively; the total number of BMCs from long bones was 14.2 ± 4.2 × 108 cells (Table 1). In contrast, the number of BMCs harvested by the conventional aspiration method was 7.7 ± 4.1 × 108 cells/total × 1.9 ± 1.0 × 107 cells/ml). This indicates that a significantly greater number of BMCs was harvested by the perfusion method than by the conventional aspiration method. The perfusion method was performed a total of 42 times using 15 cynomolgus monkeys, and no complications such as pulmonary infarction or paralysis were observed. Furthermore, the difficulty of collecting BMCs from the thin iliac crest of the cynomolgus monkeys was also overcome by using this method.

Table Table 1.. Number of BMCs in long bone
BoneNumber of BMCs
  1. a

    The perfusion method was performed 42 times using 15 monkeys.

Humerus4.3 ± 1.4 × 108 cells
Femur2.7 ± 0.6 × 108 cells
Tibia0.2 ± 0.1 × 108 cells
Total14.2 ± 4.2×108 cells

Analyses of Cell Surface Antigens and Colony-Forming Assay

We examined the percentage of T cells in the BMCs collected by both the conventional aspiration method and perfusion method. As shown in Table 2, there were more than 40% of T cells (CD4+ and CD8+ cells) in the PBMNCs and more than 20% T cells in the BMCs collected using the conventional aspiration method, whereas there were less than 6% T cells in those collected by the perfusion method. These findings indicate that the contamination with the peripheral blood, as determined by the frequency of CD4+ and CD8+ T cells, was significantly lower (p < 0.005) in the perfusion method than in the conventional method, which consists of multiple BM aspirations. Therefore, the perfusion method is useful for obtaining a large number of BMCs with a low contamination with T cells.

Table Table 2.. Analyses of cell surface antigens on BMCs in monkeys
 
Cell source**CD4CD8CD20IgMCD11bCD56
  1. a

    *Cells were stained with FITC- or PE-conjugated mAbs, and analyzed using an EIPCS-XL.

  2. b

    **The results were expressed as the mean ± SD of five monkeys.

  3. c

    ***Statistical analyses were performed by t-test: p < 0.005, perfusion method versus aspiration method.

  4. d

    PBL = peripheral blood lymphocyte.

BMCs harvested by      
Perfusion method2.2 ± 2.0***3.9 ± 3.3***3.5 ± 0.56.7 ± 1.927.1 ± 9.28.5 ± 3.9
Aspiration method10.5 ± 0.712.6 ± 0.25.1 ± 1.87.0 ± 3.422.9 ± 4.05.5 ± 2.0
PBL19.9 ± 5.122.6 ± 5.213.2 ± 7.220.3 ± 4.67.7 ± 7.03.9 ± 2.2

Progenitor cell activities were next determined by in vitro CFU-C assays. BMCs collected by the perfusion or conventional aspiration method were cultured in methylallulose containing a combination of cytokines essential for hemopoiesis (SCF, Epo, IL-3, GM-CSF, and G-CSF). BMCs collected by the perfusion method generated a significantly higher number of CFU-C than those harvested by the conventional method when assayed 14 days later (67.0 ± 4.0 versus 47.3 ± 6.0/104: p < 0.001). This indicates that the frequency of progenitor cells is higher in the BMCs collected by the perfusion method than by the conventional method.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In recent years, studies concerning organ allografts associated with allogeneic BMT have been increasingly carried out using rhesus and cynomolgus monkeys [5-8]—analyses including the mixed chimerism for transplantation tolerance, the post-transplant immune status in recipients of BM allografts, and allograft tolerance induced by donor BM cells. These studies clearly show that BMT is of great benefit in the treatment of various diseases. However, one of the problems associated with BMT is the contamination of the BMCs with T cells derived from the peripheral blood when the BMCs are collected using the multiple aspiration method. Although various treatments such as anti-T cell Ab and immunosuppressants have been used to reduce the undesirable functions of these contaminant T cells, none of those treatments were particularly effective. To minimize the T cell contamination, we developed the new “perfusion method” using cynomolgus monkeys (Fig. 1). The contamination with peripheral blood, determined by the frequency of CD4+ and CD8+ T cells, is significantly lower using this method (<6%) than using the aspiration method (>20%). Furthermore, it is of importance that the frequency of progenitor cells harvested by the perfusion method is greater than by the aspiration method, this being attributable to the relative decrease in the number of T cells contaminating the BMCs.

It has been generally established that more than 2 × 108 BMCs/kg are necessary for BMT. Therefore, multiple BM aspirations are essential for collecting BMCs; this results in a large burden on donors due to the duration of the operation. In contrast, the perfusion method is completed within 20 min after the anesthetization. Consequently, using the perfusion method, a large number of BMCs can rapidly be harvested without any complications such as pulmonary infarction or paralysis (when tested 42 times using 15 cynomolgus monkeys), thus reducing the burden on the donors.

In humans, since the number of the patients for whom BMT is indispensable has been rapidly increasing in recent years, HLA-one locus mismatched BMT or unrelated BMT have been performed, but GVHD, graft rejection, and incomplete T cell recovery have been reported [12-15]. The perfusion method, which minimizes the contamination of the BMCs with T cells from the peripheral blood, may prevent these problems, particularly GVHD. Since red BM is detected in the long bones to age 20 [16], BMCs harvested using the perfusion method from adolescent donors who are brain dead may also be possible.

We believe that this perfusion method would also contribute to gene therapy and organ allografts in conjunction with BMT even in humans.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Ms. Y. Tokuyama, Ms. M. Shinkawa, and Ms. S. Miura for their expert technical assistance, and Mr. Hilary Eastwick-Field and Ms. K. Ando for their help in the preparation of the manuscript.

This work was supported by a grant from the “Haiteku Research Center,” a grant-in-aid for scientific research (B) 11470062, and grants-in-aid for scientific research on priority areas (A) 10181225 and (A) 11162221 for the Ministry of Education, a grant from the “Millennium Project” of the Science and Technology Agency, and also a grant from Japan Immunoresearch Laboratories Co., Ltd. (JIMRO).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References