IN VITRO Vol. 11, No. 6, 1975
EFFECT O F O S M O T I C P R E S S U R E O N N E U R O G E N E S I S CULTURES OF CHICK EMBRYO SPINAL CORDS
IN
KENNETH R. S. F I S H E R / SERGEY FEDOROFF, AND ELEANOR L. WENGER 2
Department of Anatomy, University of Saskatchewan, Saskaloon, Saskatchewan, Canada SUMMARY
The osmotic pressure of the medium in stoppered, roller tube cultures increased by an average of 17 • 6 mOsM per kg of water during 3 days of incubation at 37~ irrespective of the initial osmolality (280 to 340 mOsM) of the medium. The increase was apparently due to evaporation of water from the medium into the gas phase of the roller tube. This observation led us to study the effect of osmotic pressure on neuronal differentiation in cultures of chick embryo spinal cords. Spinal cords were excised from stage 16 to 19 (2.5 to 3 days of incubation) or stage 36 (10 days) chick embryos and cultured as fragments on collagen-coated cover slips in roller tubes at 37~ for 21 days. The medium was adjusted to 283 • 3, 300 • 3, 323 • 3, or 342 4- 3 mOs~ per kg with saturated choline chloride solution or distilled water. The results indicate that the nature of the neuronal differentiation in vitro was not altered by the osmolality of the medium. The proportion of cultures containing neurons was influenced byosmolality. In the 300 + 3 mOs~ medium, 75% of all the stage 36 cultures initiated contained neurons, and 52% of all the stage 16 to 19 cultures initiated contained neurons. In the other media the proportion of neuron-containing cultures was lower. Two conclusions were drawn, l~eurogenesis in cultures of embryonic chick spinal cord fragments is sensitive to an increase in the initial osmotic pressure of the medium as small as 20 mOsM above the optimal 300 mOs~. As a result of the 17 mOsM increase which always occurred in the culture medium between feedings, the optimum osmolality for neuronal development is in fact a range, from 300 to 317 mOsM. Key words: neuron; differentiation; in vitro; chick embryo. As tissue culture evolves from an "art form" into a quantifiable scientific tool, the relationship between the culture environment and the material cultured assumes increasing importance. This is particularly true in studies of embryonic development where, in addition to maintenance of existing structure or function in vitro, biochemical and/or morphological differentiation must be obtained. The culture environment is known to affect the expression of morphogenesis in vitro (1, 2). One variable is the osmotic pressure of media and sera (3). When osmotic pressure and other factors inherent in the final culture medium vary, the fate or behavior of cultured embryonic cells or tissue becomes difficult to interpret.
This paper deals with changes in the osmotic pressure of culture media during incubation, and the response of nervous tissue from chick embryos of stages 16 to 19 and 36 (4) to culture media with various osmotic pressures. MATERIALS AND ~/[ETHODS
Preparation of cultures. Fertile, White Leghorn eggs (strain H / N ) were incubated for approximately 2.5 to 3 or 10 days at 38~ respectively, until the embryos reached stage 16 to 19, or stage 36 (4). The embryos were removed and placed ia sterile Hanks' balanced salt solution. Spinal cord segments from the level of approximately somite 6 to somite 25, of stage 16 to 19 chick embryo neural tubes were dissected. These spinal cord 1 To whom reprint requests should be sent. segments were divided into three equal parts for 2 Present address: Department of Biology, University of Saskatchewan, Saskatoon, Sask., culturing on collagen-coated (5), l l • 22-ram cover slips. Thus, each culture consisted of a Canada. 329
330
FISHER, FEDOROFF, AND WENGER
fragment of neural tube equivalent in length to about 6 or 7 somites. Cultures of stage 36 chick embryo spinal cords were made up of two transverse sections, each 0.4-mm long, from the brachial level of the spinal cord, placed on a collagen-coated l l • 22-mm cover slip. The cover slips, with spinal cord pieces, were put into test tubes (16 • 150 mm, Pyrex, rimless) and 0.7 ml of culture medium was added. The tubes were sealed with silicone rubber stoppers and incubated at 37~ For the first 24 hr they were held in a static, horizontal position, then they were transferred to a roller drum which revolved at 12 revolutions per hr. The culture medium was replaced twice a week. Culture medium. The culture medium consisted of a mixture of 61 ml of medium 199, 32 ml of horse serum, 5 ml of 9-day chick embryo extract (1:1 in Hanks' saline), and 3.2 ml of glucose solution (20%), to make the final concentration of glucose in the medium 60 mg per ml. The pH of this medium was adjusted to 7.9 • 0.1 (cherry red color of phenol red) with a few drops of autoclaved 1.4% NaHCO.~. The osmolality was adjusted to 300 • 3 mOsM, with either distilled water or saturated choline chloride (6) solution. Determination of osmotic pressure. Throughout the present study an "Osmette" (Precision Systems Ltd.) was used to measure (by freezing point depression) the osmotic pressure of the medium. During the early phase of this work, media were pooled in order to get the 2.0 ml required for measurement. Later, micro-attachments requiring only 0.2 ml were used, permitting two samples from each roller tube to be used for osmotic pressure measurement. The osmometer was calibrated before and after each set of measurements by means of standard solutions (Advanced Instruments Inc., I000 Highland Ave., Needham Heights, Mass., U.S.A.; in Canada, "Canlab"). For each measurement of the osmolality of medium, at least four to six determinations were made. Four types of media differing only in osmolalities were used; 280 to 286, 300 to 306, 320 to 326, or 340 to 343 mOsM. Distilled water or saturated choline chloride (6) solution was used to make the adjustment. These media will be referred to as medium 280, 300, 320, and 340 for purposes of simplicity. The osmotic pressure of the medium was measured before, during, and after the 3-day period between culture feedings.
Analysis of cultures. After 21 days in vitro all cultures were fixed in Cajal's formalin-ammonium bromide fixative and stained by modified Bodian Protargol silver technique (7). The cultures were examined microscopically for evidence of neuronal differentiation. Neurons were considered to be those cells which possessed argyrophilic fibrils in their processes and perikarya (8), and had large nuclei with nucleoli, considerable amounts of cytoplasm, and numerous processes. RESULTS Stage 16 to 19 cultures, after 21 days in vitro, contained numerous multipolar nerve cell bodies throughout the zone of outgrowth (Figs. 3 and 4), indicating that at this stage the nerve cells maintained their migrating ability (9). In this zone non-neuronal cells formed a monolayer and neurites grew out from the nerve cell bodies forming a feltlike meshwork of fibrils (Figs. 3, 4, and 5). After 21 days in vitro, stage 36 cultures consisted of a monolayer of non-neuronal cells whose nuclei were slightly larger and morphologically more heterogeneous than in stage 16 to 19 cultures (Figs. 1, 2, 4, and 6). Neurites grew out of the explant over the non-neuronal cells in parallel arrays or bundles (Fig. 2). The fascicules broke up near the edge of the zone of outgrowth as the neurites formed complex meshworks (Figs. 2 and 6). Multipolar nerve cell perikarya were only rarely found away from the explant. This was interpreted as indicating that the migratory activity of the neurons was lost by stage 36. Further studies of the effect of embryonic stage on the fate of chick embryo spinal cord cultures are in progress.
Determination of osmolality during culturing period. To determine whether or not there is a change in the osmotic pressure of the medium during the culturing period, the osmotic pressure was determined in the media from the cultures before and after incubating them for 3 days. Four types of media, with osmotic pressures of 280, 320, 340, and 342 were used. After 3 days in vitro the osmotic pressure of all media had increased by an average of 17 i 6 mOsM (Table 1). The initial osmolality of the medium had no significant effect on the magnitude of the increase (Table 1). To determine which, if any, of the culture components was responsible for the increase, the
Fro. 1. Photomicrograph of an "undifferentiated" stage 17 chick embryo spinal cord culture, 21 days in vitro in 300 mOsM medium. Despite the extensive outgrowth of nonneuronal cells, there are no nerve cell perikarya or neuronal fibers present. Bodian silver stained. X40. FIG. 2. Photomicrograph of a 21-day culture of a piece of stage 36 chick spinal cord. The major outgrowth of neuronal fibers, fasciculated near the explant, then becoming randomly oriented towards the periphery of the culture, is characteristic of well differentiated stage 36 cultures. Bodian silver stained. >
EFFECT O F O S M O T I C P R E S S U R E O N N E U R O G E N E S I S CULTURES OF CHICK EMBRYO SPINAL CORDS
IN
KENNETH R. S. F I S H E R / SERGEY FEDOROFF, AND ELEANOR L. WENGER 2
Department of Anatomy, University of Saskatchewan, Saskaloon, Saskatchewan, Canada SUMMARY
The osmotic pressure of the medium in stoppered, roller tube cultures increased by an average of 17 • 6 mOsM per kg of water during 3 days of incubation at 37~ irrespective of the initial osmolality (280 to 340 mOsM) of the medium. The increase was apparently due to evaporation of water from the medium into the gas phase of the roller tube. This observation led us to study the effect of osmotic pressure on neuronal differentiation in cultures of chick embryo spinal cords. Spinal cords were excised from stage 16 to 19 (2.5 to 3 days of incubation) or stage 36 (10 days) chick embryos and cultured as fragments on collagen-coated cover slips in roller tubes at 37~ for 21 days. The medium was adjusted to 283 • 3, 300 • 3, 323 • 3, or 342 4- 3 mOs~ per kg with saturated choline chloride solution or distilled water. The results indicate that the nature of the neuronal differentiation in vitro was not altered by the osmolality of the medium. The proportion of cultures containing neurons was influenced byosmolality. In the 300 + 3 mOs~ medium, 75% of all the stage 36 cultures initiated contained neurons, and 52% of all the stage 16 to 19 cultures initiated contained neurons. In the other media the proportion of neuron-containing cultures was lower. Two conclusions were drawn, l~eurogenesis in cultures of embryonic chick spinal cord fragments is sensitive to an increase in the initial osmotic pressure of the medium as small as 20 mOsM above the optimal 300 mOs~. As a result of the 17 mOsM increase which always occurred in the culture medium between feedings, the optimum osmolality for neuronal development is in fact a range, from 300 to 317 mOsM. Key words: neuron; differentiation; in vitro; chick embryo. As tissue culture evolves from an "art form" into a quantifiable scientific tool, the relationship between the culture environment and the material cultured assumes increasing importance. This is particularly true in studies of embryonic development where, in addition to maintenance of existing structure or function in vitro, biochemical and/or morphological differentiation must be obtained. The culture environment is known to affect the expression of morphogenesis in vitro (1, 2). One variable is the osmotic pressure of media and sera (3). When osmotic pressure and other factors inherent in the final culture medium vary, the fate or behavior of cultured embryonic cells or tissue becomes difficult to interpret.
This paper deals with changes in the osmotic pressure of culture media during incubation, and the response of nervous tissue from chick embryos of stages 16 to 19 and 36 (4) to culture media with various osmotic pressures. MATERIALS AND ~/[ETHODS
Preparation of cultures. Fertile, White Leghorn eggs (strain H / N ) were incubated for approximately 2.5 to 3 or 10 days at 38~ respectively, until the embryos reached stage 16 to 19, or stage 36 (4). The embryos were removed and placed ia sterile Hanks' balanced salt solution. Spinal cord segments from the level of approximately somite 6 to somite 25, of stage 16 to 19 chick embryo neural tubes were dissected. These spinal cord 1 To whom reprint requests should be sent. segments were divided into three equal parts for 2 Present address: Department of Biology, University of Saskatchewan, Saskatoon, Sask., culturing on collagen-coated (5), l l • 22-ram cover slips. Thus, each culture consisted of a Canada. 329
330
FISHER, FEDOROFF, AND WENGER
fragment of neural tube equivalent in length to about 6 or 7 somites. Cultures of stage 36 chick embryo spinal cords were made up of two transverse sections, each 0.4-mm long, from the brachial level of the spinal cord, placed on a collagen-coated l l • 22-mm cover slip. The cover slips, with spinal cord pieces, were put into test tubes (16 • 150 mm, Pyrex, rimless) and 0.7 ml of culture medium was added. The tubes were sealed with silicone rubber stoppers and incubated at 37~ For the first 24 hr they were held in a static, horizontal position, then they were transferred to a roller drum which revolved at 12 revolutions per hr. The culture medium was replaced twice a week. Culture medium. The culture medium consisted of a mixture of 61 ml of medium 199, 32 ml of horse serum, 5 ml of 9-day chick embryo extract (1:1 in Hanks' saline), and 3.2 ml of glucose solution (20%), to make the final concentration of glucose in the medium 60 mg per ml. The pH of this medium was adjusted to 7.9 • 0.1 (cherry red color of phenol red) with a few drops of autoclaved 1.4% NaHCO.~. The osmolality was adjusted to 300 • 3 mOsM, with either distilled water or saturated choline chloride (6) solution. Determination of osmotic pressure. Throughout the present study an "Osmette" (Precision Systems Ltd.) was used to measure (by freezing point depression) the osmotic pressure of the medium. During the early phase of this work, media were pooled in order to get the 2.0 ml required for measurement. Later, micro-attachments requiring only 0.2 ml were used, permitting two samples from each roller tube to be used for osmotic pressure measurement. The osmometer was calibrated before and after each set of measurements by means of standard solutions (Advanced Instruments Inc., I000 Highland Ave., Needham Heights, Mass., U.S.A.; in Canada, "Canlab"). For each measurement of the osmolality of medium, at least four to six determinations were made. Four types of media differing only in osmolalities were used; 280 to 286, 300 to 306, 320 to 326, or 340 to 343 mOsM. Distilled water or saturated choline chloride (6) solution was used to make the adjustment. These media will be referred to as medium 280, 300, 320, and 340 for purposes of simplicity. The osmotic pressure of the medium was measured before, during, and after the 3-day period between culture feedings.
Analysis of cultures. After 21 days in vitro all cultures were fixed in Cajal's formalin-ammonium bromide fixative and stained by modified Bodian Protargol silver technique (7). The cultures were examined microscopically for evidence of neuronal differentiation. Neurons were considered to be those cells which possessed argyrophilic fibrils in their processes and perikarya (8), and had large nuclei with nucleoli, considerable amounts of cytoplasm, and numerous processes. RESULTS Stage 16 to 19 cultures, after 21 days in vitro, contained numerous multipolar nerve cell bodies throughout the zone of outgrowth (Figs. 3 and 4), indicating that at this stage the nerve cells maintained their migrating ability (9). In this zone non-neuronal cells formed a monolayer and neurites grew out from the nerve cell bodies forming a feltlike meshwork of fibrils (Figs. 3, 4, and 5). After 21 days in vitro, stage 36 cultures consisted of a monolayer of non-neuronal cells whose nuclei were slightly larger and morphologically more heterogeneous than in stage 16 to 19 cultures (Figs. 1, 2, 4, and 6). Neurites grew out of the explant over the non-neuronal cells in parallel arrays or bundles (Fig. 2). The fascicules broke up near the edge of the zone of outgrowth as the neurites formed complex meshworks (Figs. 2 and 6). Multipolar nerve cell perikarya were only rarely found away from the explant. This was interpreted as indicating that the migratory activity of the neurons was lost by stage 36. Further studies of the effect of embryonic stage on the fate of chick embryo spinal cord cultures are in progress.
Determination of osmolality during culturing period. To determine whether or not there is a change in the osmotic pressure of the medium during the culturing period, the osmotic pressure was determined in the media from the cultures before and after incubating them for 3 days. Four types of media, with osmotic pressures of 280, 320, 340, and 342 were used. After 3 days in vitro the osmotic pressure of all media had increased by an average of 17 i 6 mOsM (Table 1). The initial osmolality of the medium had no significant effect on the magnitude of the increase (Table 1). To determine which, if any, of the culture components was responsible for the increase, the
Fro. 1. Photomicrograph of an "undifferentiated" stage 17 chick embryo spinal cord culture, 21 days in vitro in 300 mOsM medium. Despite the extensive outgrowth of nonneuronal cells, there are no nerve cell perikarya or neuronal fibers present. Bodian silver stained. X40. FIG. 2. Photomicrograph of a 21-day culture of a piece of stage 36 chick spinal cord. The major outgrowth of neuronal fibers, fasciculated near the explant, then becoming randomly oriented towards the periphery of the culture, is characteristic of well differentiated stage 36 cultures. Bodian silver stained. >
