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Human bone marrow colony formation in diffusion chambers
Printed in Sweden Copyright Q 1978 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/78/1111-021~2.00/0
Experimental
HUMAN
BONE
Ceil Research 111 (1978) 2 19-222
MARROW
COLONY
IN DIFFUSION
FORMATION
CHAMBERS
Normal
Values
M. Y. GORDON and I. D. C. DOUGLAS Department
of Biophysics,
Institute
of Cancer
Research,
Sutton,
Surrey,
UK
SUMMARY Two-hundred and twentylive marrow samples, aspirated from the iliac crests of hematologically normal individuals, have been assessed for colony formation in agar in diffusion chambers. These assays were made over a period of 30 months and the results show that the mean, range and distribution of normal values has remained the same during this time.
Comparative studies of human bone marrow function, using colony formation as an endpoint, require that all samples be stimulated to the same extent and that the culture conditions be rigorously controlled. The problem of maintaining consistent culture conditions for human bone marrow colony growth in vitro derives from two main factors. (1) The lack of a standard to monitor the efficiency of a culture system with time, since each normal individual’s marrow contains a different number of colony forming cells; (2) the vagaries experienced in the production of the colonystimulating activity (CSA) required for the formation of bone marrow colonies in vitro. One test of the reproducibility of a human marrow colony assay would be to compare any fluctuations in the means, ranges and distributions of normal marrow colony yields over a long period of time. This re1 Present address: Department of Haematology, Lukes Hospital, Guildford, Surrey, UK.
St
port gives such data, which were obtained using an agar diffusion chamber colony assay. Over a period of 30 months the samples showed a wide range of colony-forming abilities but the mean values, as well as the ranges and distributions, were consistent during the time investigated.
MATERIALS
AND
METHODS
The 225 marrow samples used for the present series were obtained over a 30 month period from October 1974 to March 1977. All of these samples were considered to be hematologically normal on conventional morphological grounds. Two 5 ml of marrow were aspirated from the iliac crests of the donors and the nucleated cells separated by sedimentation at 1 g. The cells were thrice washed and assayed for their colony-forming ability according to the agar diffusion chamber technique described by Gordonkt al. [l]. Briefly, the cells were suspended in agar medium and introduced into modified diffusion chambers. The sealed chambers were inserted into the peritoneal cavities of irradiated (900 rad 60Co y rays) C57Bl mice. Seven to 8 days later the numbers of colonies present in the agar clot were counted. Only aggregates of more than 50 cells were scored as colonies and these contained mainly granulocyte cells with some mononuclear cells [ 11. Exp Cell
Rev I1 I (I 978)
220
Gordon and Douglas
.
terial for other hematological of 5 marrow samples were month period at intervals of the data in table 1 show up ference in the incidence of cells in these aspirates.
tests. A total taken over a 5 1-12 weeks and to a 2-fold difcolony-forming
:
DISCUSSION Colony assays can only be used for comparative assessment of human bone marrow function providing the normal range of colony-forming ability is known and the culture conditions are sufficiently reproducible to maintain this range over long periods of time. This is particularly important when an individual’s marrow function is monitored during disease or treatment. 1975 1976 1977 1974 Ja”-Mar act-Dee (4, sample*) c77 wmpkr, c72 salnplell) cm sample.) The agar diffusion chamber method has been proposed as a simple alternative to Fig. 1. Abscissa: samples; ordinate: colonies/2 x 105 bone marrow cells. the in vitro culture of human bone marrow Colony yields from 216 normal marrow aspirates. in semi-solid media [ 11. Furthermore, analEach point represents a different individual. Horizontal bars, mean colony yield for each period shown. ysis of results from colony assays of murine bone marrow, both in agar in vitro and in diffusion chambers, showed that the diffusion chamber technique produced the RESULTS most consistent values [2]. Fig. 1 shows the individual colony yields for This technique eliminates the use of any 216 of the marrow samples assayed in this conventional source of colony stimulating series. The range for the whole series was activity (CSA) from the culture system, 2.5-75 colonies/2x lo5 cells cultured. Both thus removing one source of variation exthe means and ranges of colony-forming ability were similar at the four intervals investigated. Nine further normal marrow Table 1. The variation in the incidence of samples were assayed during this period: colony forming cells in the marrow of a these produced no colonies in culture giving single individual a failure rate of approx. 4 %. No. of colonies/ Date sample It was possible also to investigate the ex- taken 2x 105 bone marrow cells tent of variation in the colony-forming abili819 1976 26.5 ty within the normal individual at different 3019 1976 21.9 times. Unusual circumstances allowed us to S/l0 1976 16.7 l/2 1977 23.6 use samples aspirated from a single normal 2812 1977 36.2 donor whose cells were taken as target ma-
Exp Cell Res III(1978)
Human
perienced in vitro. We have endeavoured also to control other factors which may influence the measured colony-forming ability of human marrow aspirates. All samples were taken from the iliac crests as the incidence of colony-forming cells has been shown to vary in different parts of the skeleton. The distribution of active marrow around the pelvis has, however, been found to be uniform [3]. Furthermore, the volume of the samples was restricted to between 2 and 5 ml, since extreme variation in the amount aspirated results in more or less dilution of the samples by peripheral blood [4]. The present results show that the means and ranges of normal human marrow colony-forming ability were similar over a period of 30 months, indicating that the conditions for colony growth were consistent. The failure rate of approx. 4% compares favourably with the figure of 30-40% quoted by Douglas & Pickering [5] for in vitro culture. The data agree with the findings of many others who, using in vitro systems, find a wide range of colony yields from normal human marrow [6] and with our experience of colony yields from 9+t/2 x 105 cells using leukocyte feeder layers as a source of CSA. Similarly, the variation within the individual over a period of 5 months was of the same order as that described by Entringer et al. [6]. However, there appear to be no other reports comparing normal values over a long period of time, although this may be one valid test of the consistency of the culture conditions. The elimination of CSA from the culture system used here appears to be advantageous in maintaining constant culture conditions. Douglas & Pickering [5] have investigated recently the variability of different sources of CSA and found that the
bone marrow
colony formation
221
effective sources were not consistently better or worse than one another, even when several types and batches of CSA were tested against a single marrow. In contrast, Entringer et al. [6] did not find any significant variation in colony yield when cells from a single individual were plated over different white blood cell feeder layers. It is not certain what factors are responsible for the induction of colony formation in diffusion chambers. An indirect stimulation by the mouse may activate colony-stimulating cells in the cultured marrow although there is no evidence that monocytic cells play a part in colony formation in this system [l]. Although an inhibitory effect of polymorphonuclear neutrophils cannot be ruled out, this has only been detected in unstimulated cultures [7, 81. Thus, it is unlikely that cellular interactions within the chambers contribute to the variability shown in fig. 1. The data described in this communication define the range of normal values for the colony-forming ability of human marrow in agar diffusion chambers and provides further evidence that differing numbers of colony-forming cells in aspirated marrow are the major contribution to the wide range of normal values seen in culture [6,8]. The results we have obtained from normal human bone marrow were consistent over a period of 30 months. This finding indicates that the diffusion chamber technique complies with the basic requirements for making long term studies of the colonyforming abilities of normal and abnormal human marrow. However, as pointed out by others [6] the wide range of normal values makes comparison between normal and abnormal individuals difficult and emphasises the necessity for studying large numbers of samples. Exp Cc// Res III
(1978)
222
Gordon and Douglas
The authors acknowledge the support provided by the NC1 and wish to thank B. M. J. Pickering and Miss V. B. Shepherd for their excellent technical assistance.
REFERENCES 1. Gordon, M Y, Blackett, N M & Douglas, I DC, Brit j haematol31 (1975) 103. 2. Blackett, N M, Marsh, J C, Gordon, M Y, Okell, S F & Aguado, M, Exp haematol(1977). In press. 3. Gordon, M Y, Douglas, I D C, Clink, H M & Pickering, B M J, Brit j haemato132 (1976) 537. 4. Gordon, M Y & Douglas, I D C, Exp haematol 5 (1977) 274.
Exp Cell
Res I I I (1978)
5. Douglas, I D C & Pickering, B M J, Exp cell res 104 (1977) 95. 6. Entringer, M A, Robinson, W A & Kumick, J E, Exp haematol5 (1977) 125. 7. Broxmeyer, H E, Mendelsohn, N & Moore, M A S, Leukaemia res 1 (1977) 3. 8. Moore, M A S, Burgess, A W, Metcalf, D, McCulloch, E A, Robinson, W A, Dicke, K A, Chervenick, P A, Buk, J M, Wu, A M, Stanley, E R, Goldman, J & Testa, N, Br j cancer 35 (1977) 500. Received May 27, 1977 Revised version received July 25, 1977 Accepted July 28, 1977
Experimental
HUMAN
BONE
Ceil Research 111 (1978) 2 19-222
MARROW
COLONY
IN DIFFUSION
FORMATION
CHAMBERS
Normal
Values
M. Y. GORDON and I. D. C. DOUGLAS Department
of Biophysics,
Institute
of Cancer
Research,
Sutton,
Surrey,
UK
SUMMARY Two-hundred and twentylive marrow samples, aspirated from the iliac crests of hematologically normal individuals, have been assessed for colony formation in agar in diffusion chambers. These assays were made over a period of 30 months and the results show that the mean, range and distribution of normal values has remained the same during this time.
Comparative studies of human bone marrow function, using colony formation as an endpoint, require that all samples be stimulated to the same extent and that the culture conditions be rigorously controlled. The problem of maintaining consistent culture conditions for human bone marrow colony growth in vitro derives from two main factors. (1) The lack of a standard to monitor the efficiency of a culture system with time, since each normal individual’s marrow contains a different number of colony forming cells; (2) the vagaries experienced in the production of the colonystimulating activity (CSA) required for the formation of bone marrow colonies in vitro. One test of the reproducibility of a human marrow colony assay would be to compare any fluctuations in the means, ranges and distributions of normal marrow colony yields over a long period of time. This re1 Present address: Department of Haematology, Lukes Hospital, Guildford, Surrey, UK.
St
port gives such data, which were obtained using an agar diffusion chamber colony assay. Over a period of 30 months the samples showed a wide range of colony-forming abilities but the mean values, as well as the ranges and distributions, were consistent during the time investigated.
MATERIALS
AND
METHODS
The 225 marrow samples used for the present series were obtained over a 30 month period from October 1974 to March 1977. All of these samples were considered to be hematologically normal on conventional morphological grounds. Two 5 ml of marrow were aspirated from the iliac crests of the donors and the nucleated cells separated by sedimentation at 1 g. The cells were thrice washed and assayed for their colony-forming ability according to the agar diffusion chamber technique described by Gordonkt al. [l]. Briefly, the cells were suspended in agar medium and introduced into modified diffusion chambers. The sealed chambers were inserted into the peritoneal cavities of irradiated (900 rad 60Co y rays) C57Bl mice. Seven to 8 days later the numbers of colonies present in the agar clot were counted. Only aggregates of more than 50 cells were scored as colonies and these contained mainly granulocyte cells with some mononuclear cells [ 11. Exp Cell
Rev I1 I (I 978)
220
Gordon and Douglas
.
terial for other hematological of 5 marrow samples were month period at intervals of the data in table 1 show up ference in the incidence of cells in these aspirates.
tests. A total taken over a 5 1-12 weeks and to a 2-fold difcolony-forming
:
DISCUSSION Colony assays can only be used for comparative assessment of human bone marrow function providing the normal range of colony-forming ability is known and the culture conditions are sufficiently reproducible to maintain this range over long periods of time. This is particularly important when an individual’s marrow function is monitored during disease or treatment. 1975 1976 1977 1974 Ja”-Mar act-Dee (4, sample*) c77 wmpkr, c72 salnplell) cm sample.) The agar diffusion chamber method has been proposed as a simple alternative to Fig. 1. Abscissa: samples; ordinate: colonies/2 x 105 bone marrow cells. the in vitro culture of human bone marrow Colony yields from 216 normal marrow aspirates. in semi-solid media [ 11. Furthermore, analEach point represents a different individual. Horizontal bars, mean colony yield for each period shown. ysis of results from colony assays of murine bone marrow, both in agar in vitro and in diffusion chambers, showed that the diffusion chamber technique produced the RESULTS most consistent values [2]. Fig. 1 shows the individual colony yields for This technique eliminates the use of any 216 of the marrow samples assayed in this conventional source of colony stimulating series. The range for the whole series was activity (CSA) from the culture system, 2.5-75 colonies/2x lo5 cells cultured. Both thus removing one source of variation exthe means and ranges of colony-forming ability were similar at the four intervals investigated. Nine further normal marrow Table 1. The variation in the incidence of samples were assayed during this period: colony forming cells in the marrow of a these produced no colonies in culture giving single individual a failure rate of approx. 4 %. No. of colonies/ Date sample It was possible also to investigate the ex- taken 2x 105 bone marrow cells tent of variation in the colony-forming abili819 1976 26.5 ty within the normal individual at different 3019 1976 21.9 times. Unusual circumstances allowed us to S/l0 1976 16.7 l/2 1977 23.6 use samples aspirated from a single normal 2812 1977 36.2 donor whose cells were taken as target ma-
Exp Cell Res III(1978)
Human
perienced in vitro. We have endeavoured also to control other factors which may influence the measured colony-forming ability of human marrow aspirates. All samples were taken from the iliac crests as the incidence of colony-forming cells has been shown to vary in different parts of the skeleton. The distribution of active marrow around the pelvis has, however, been found to be uniform [3]. Furthermore, the volume of the samples was restricted to between 2 and 5 ml, since extreme variation in the amount aspirated results in more or less dilution of the samples by peripheral blood [4]. The present results show that the means and ranges of normal human marrow colony-forming ability were similar over a period of 30 months, indicating that the conditions for colony growth were consistent. The failure rate of approx. 4% compares favourably with the figure of 30-40% quoted by Douglas & Pickering [5] for in vitro culture. The data agree with the findings of many others who, using in vitro systems, find a wide range of colony yields from normal human marrow [6] and with our experience of colony yields from 9+t/2 x 105 cells using leukocyte feeder layers as a source of CSA. Similarly, the variation within the individual over a period of 5 months was of the same order as that described by Entringer et al. [6]. However, there appear to be no other reports comparing normal values over a long period of time, although this may be one valid test of the consistency of the culture conditions. The elimination of CSA from the culture system used here appears to be advantageous in maintaining constant culture conditions. Douglas & Pickering [5] have investigated recently the variability of different sources of CSA and found that the
bone marrow
colony formation
221
effective sources were not consistently better or worse than one another, even when several types and batches of CSA were tested against a single marrow. In contrast, Entringer et al. [6] did not find any significant variation in colony yield when cells from a single individual were plated over different white blood cell feeder layers. It is not certain what factors are responsible for the induction of colony formation in diffusion chambers. An indirect stimulation by the mouse may activate colony-stimulating cells in the cultured marrow although there is no evidence that monocytic cells play a part in colony formation in this system [l]. Although an inhibitory effect of polymorphonuclear neutrophils cannot be ruled out, this has only been detected in unstimulated cultures [7, 81. Thus, it is unlikely that cellular interactions within the chambers contribute to the variability shown in fig. 1. The data described in this communication define the range of normal values for the colony-forming ability of human marrow in agar diffusion chambers and provides further evidence that differing numbers of colony-forming cells in aspirated marrow are the major contribution to the wide range of normal values seen in culture [6,8]. The results we have obtained from normal human bone marrow were consistent over a period of 30 months. This finding indicates that the diffusion chamber technique complies with the basic requirements for making long term studies of the colonyforming abilities of normal and abnormal human marrow. However, as pointed out by others [6] the wide range of normal values makes comparison between normal and abnormal individuals difficult and emphasises the necessity for studying large numbers of samples. Exp Cc// Res III
(1978)
222
Gordon and Douglas
The authors acknowledge the support provided by the NC1 and wish to thank B. M. J. Pickering and Miss V. B. Shepherd for their excellent technical assistance.
REFERENCES 1. Gordon, M Y, Blackett, N M & Douglas, I DC, Brit j haematol31 (1975) 103. 2. Blackett, N M, Marsh, J C, Gordon, M Y, Okell, S F & Aguado, M, Exp haematol(1977). In press. 3. Gordon, M Y, Douglas, I D C, Clink, H M & Pickering, B M J, Brit j haemato132 (1976) 537. 4. Gordon, M Y & Douglas, I D C, Exp haematol 5 (1977) 274.
Exp Cell
Res I I I (1978)
5. Douglas, I D C & Pickering, B M J, Exp cell res 104 (1977) 95. 6. Entringer, M A, Robinson, W A & Kumick, J E, Exp haematol5 (1977) 125. 7. Broxmeyer, H E, Mendelsohn, N & Moore, M A S, Leukaemia res 1 (1977) 3. 8. Moore, M A S, Burgess, A W, Metcalf, D, McCulloch, E A, Robinson, W A, Dicke, K A, Chervenick, P A, Buk, J M, Wu, A M, Stanley, E R, Goldman, J & Testa, N, Br j cancer 35 (1977) 500. Received May 27, 1977 Revised version received July 25, 1977 Accepted July 28, 1977