Development of Tissue Culture Techniques and Hardware to Study Mineralization under Microgravity Conditions.

J.J.W.A. van Loon¹, J.P. Veldhuijzen¹, E.J. Windgassen², T. Brouwer³, K. Wattel³, M. van Vilsteren³ and P. Maas⁴.

Academic Centre for Dentistry Amsterdam (ACTA), Dept. of Oral Cell Biology¹ and Dept. of Engineering² and Vrije Universiteit, Fac. of Biology, Dept. of Electronics³, and of Engineering⁴. Amsterdam, The Netherlands.

To study the effects of weightlessness on fetal mouse long bone rudiment growth and mineralization we have developed a tissue culture system for the Biorack facility of Spacelab. The technique uses standard liquid tissue culture medium, supplemented with Na-b-glycerophosphate, confined in gas permeable polyethylene bags mounted inside ESA Biorack Type-I experiment containers. The containers can be flushed with an air/5% CO₂ gas mixture necessary for the physiological bicarbonate buffer used. Small amounts of fluid can be introduced at the beginning (e.g. radioactive labels for incorporation studies) or at the end of the experiment (fixatives). A certain form of mechanical stimulation (continuous compression) can be used to counteract the, possibly, adverse effect of microgravity. Using 16 day old metatarsals the in vitro calcification process under microgravity conditions can be studied for a 4 day period.

Key words: bone, weightlessness, in vitro, mineralization, mechanical stimulation, bicarbonate buffer, spacelab.

Published in: Advances in Space Research, 14(8), 289-298, 1994.

It is well known that the mechanical environment is an important factor in the development and maintenance of skeletal tissues. Increased mechanical stress on bone tissue changes the bone density and morphology, resulting in an increased bone mass.⁽²⁾ A low mechanical stress environment leads to a rapid bone loss as is demonstrated in immobilization⁽¹⁵⁾ and bedrest⁽¹¹⁾ studies. In various experimental set-ups such as tail suspension^(1,5) or under actual microgravity⁽¹²⁾ during space flight, a decrease in bone mass is reported which is related to a decreased mineral deposition.

There is a very limited number of reports on a change in direct effects of osteoclastic bone resorption under actual microgravity conditions.^(4,6,7,19) Whether the effects on bone cells in vivo are indeed caused directly by a changed mechanical stress regime or that the response of bone is the result of a changed amount of circulating hormones which act on bone cell metabolism (such as corticosteroids or 24,25(OH)₂D), is not clear.⁽¹³⁾

To exclude effects of systemic factors, we are using an in vitro model to study the effect of mechanical loading on skeletal tissues. We have shown that intermittent (IC) and continuous compression (CC) of 13 kPa above ambient increases mineralization in 16 days old fetal mouse long bone rudiments and calvaria,^(8,10) whereas in 17 day old metatarsals and calvaria resorption of the mineralized matrix by osteoclasts was decreased.^(3,8) It is hypothesized that non-compressed control bones, which show a decreased mineralization and an increased resorption, resemble a situation of disuse. To examine if microgravity is an even more extreme situation of disuse it is tempting to study the reaction of these fetal metatarsal long bone rudiments cultured under microgravity conditions with and without compression. In order to conduct such an experiment in the Biorack facility of Spacelab we have developed a culture system and the appropriate hardware to be used in the standard Biorack Type-I containers. This experiment, 02-NL Bones, is planned to fly on board the Space Shuttle during the first International Microgravity Laboratory (IML-1) mission, STS-42. This paper describes the organ culture techniques and hardware developed for an in vitro study with fetal mouse long bone rudiments under microgravity with or without compression. The same techniques seem also applicable for in vitro studies of other mammalian organs, tissues and cells under microgravity.

Fig. 2.1. Opened Type-I/E container (20´40´80 mm) with experiment hardware fitted to the lid. a: one way valve for introducing the gas mixture, b: holes for ample flushing the gas mixture between the culture bags, c: connector to Biorack power and data lines.

2.2.1 Long Bone Rudiments

16 Day old foetuses of Swiss albino mice were used. The metatarsals (average dimensions: length 1.2 mm, diameter 0.4 mm) are carefully dissected and cleared from adhering tissues without disturbing the perichondrium and rinsed in Puck's buffer G (Gibco, Paisley, Scotland).

Fig. 2.2. Details of experiment hardware inside a Type-I/E container. a: pressure sensor, b: glass ampoule, c: metal spline.

2.2.2 Standard Laboratory Procedures

Rudiments are cultured up to 4 or 5 days in standard 24 well tissue culture plates (Greiner) in 300 ml of aMEM medium without nucleosides (Gibco), supplemented with 10% heat inactivated normal rat serum (TNO, The Netherlands) and 100 units/ml penicillin/streptomycin (Difco). To sustain good mineralization an organic phosphate source, 0-2.0 mM Na-b-glycerophosphate (Sigma) is used.^(16,17) Control rudiments are cultured in a standard air/5% CO₂ incubator (37°C, 100% humidity).

Rudiments cultured under continuous compression (CC) are placed in a stainless steel culture chamber (37°C, air/5% CO₂,100% humidity) in which the gas phase is compressed at 13 kPa above ambient. Rudiments cultured under CC are exposed in vitro to a hydrostatic compression in the same order of magnitude as the calculated muscle force acting upon these rudiments in vivo.⁽¹⁰⁾

The 16 day old rudiments, which are not calcified at the beginning of the experiment, calcify during the culture period. Determination of the length of the calcified zone (diaphysis) is used as a measure for the mineralization process.

2.2.3 Hardware and Procedures

The experiments in Biorack will be performed in Type-I containers (dimensions: 2´4´8 cm, Fig. 2.1). We use a modified version of the hardware described by Richoilley and Tixador.^(14,18) Basically, the rudiments are cultured in bags composed of two layers of gas permeable polyethylene (40´20´2 mm).

The bags are filled with 670 ml culture medium (same components as in standard laboratory conditions) and include a glass ampoule containing 25 ml liquid: e.g. radioactive labels or fixative (Fig. 2.2). In the long side of the containers a small stainless steel one-way valve is mounted in order to introduce a gas mixture of air/5% CO₂ for flushing and pressurization (Fig. 2.1a). Containers can accommodate 16 culture bags, each bag containing one long bone rudiment.

Fig. 2.3. Major parts and connections of the hardware for the Biorack experiment Bones-02NL. a: Type-I/E container, b: syringe for pressurization, c: PROD, d: pressure bottle with needle valve containing air/5%CO₂, e: connection to the Biorack power and data lines.

To release the content of the ampoule into the culture medium they are broken, by the astronauts, at a predetermined place by turning a spline situated between the culture bags (Fig. 2.2b, 2.2c). In the sides and the cover plates of the hardware, holes are made to provide ample gas flow during flushing (Fig. 2.1b).

The gas mixture of air/5% CO₂ is contained in a 150 ml stainless steel pressure bottle with needle valve (Whitey, Ohio) pressurized at 8.0×10⁶ Pa (80 bar) (Fig. 2.3d). This volume is sufficient to serve an experiment of 4 days in which eight containers are flushed and pressurized 2 times. In containers which are pressurized (CC= continuous compression; 113 kPa) a volume equivalent to 4 culture bags is devoted to accommodate newly developed electronics. These include a pressure sensor (Druck Ltd., UK; Fig. 2.2a) and electronics to correct for the temperature difference during pressurization (which is performed outside the incubator at room temperature in the glove box) and during culture in the incubator (36°C). A special Pressure Read-Out Device (PROD) is developed to monitor the increase in pressure indicated by colored LED's (Fig. 2.3c).

Pressurization is performed by pushing down the syringe piston (Fig. 2.3b). During pressurization the PROD is connected to the power and data lines of Biorack and to the container (Fig. 2.3e, 2.3a). During pressurization at room temperature a green LED is activated when the actual pressure is such that the final pressure, when placing the containers into the 36°C incubator, will be 113 ± 0.5 kPa. The actual pressure read-out via the Biorack data lines will be with an accuracy of 1 Pa.

Since both flushing and pressurization of the containers and breaking the ampoule has to be performed by the astronauts, this technique is only applicable for manned spaceflights.

2.2.4 Statistics

Results are given as mean ± standard error of the mean (SEM); significance is calculated using Student-t test statistics, 5% two-sided probability.

Table 2.I: The effect of growth (total length) and length of calcified zone (diaphysis) of 16 day old metatarsals cultured in standard tissue culture wells (MW) or polyethylene bags (bag) with (CC) or without (amb) continuous compression of 13 kPa above ambient. Media supplemented with 2.0 mM bGP. Values are mean ± SEM, *p £ 0.05.

2.3.1 Culture Bags Versus Multiwells

Since we use in our system a fluid culture medium, special precautions have to be taken.

In a series of experiments we have compared the growth and mineralization of the long bones cultured in polyethylene culture bags and in multiwells both under ambient and CC conditions (Table 2.I). It is clear from these data that the growth of the rudiments (total length at the end of the 5 day culture period) in culture bags compared to multiwells is not significantly different. CC did not affect the increase in total length in the two culture conditions investigated. We see however a significant increase in length of the mineralized diaphysis under CC conditions both in multiwells as well as in culture bags.

2.3.2 Effect of Temperature

One of the advantages in the Space Shuttle/Spacelab experiment time line is the possibility of a relatively late access for biological experiments. However, there is always a period of about 24 hours between the handover of the samples and the actual start of the experiment in Spacelab's Biorack while in orbit.

We have therefore tried to minimize biological activity during this period by keeping the experiment containers at a lower temperature than in the normal culture procedure.

Fig. 2.4 shows the effect on growth (A) and mineralization (B) after a period of 24 hours at 22°C followed by a transfer to the normal culture temperature of 37°C for 5 days. Fig. 2.4A shows that after 24 hours at 22°C, the length of the rudiment is not significantly changed and is still comparable with the starting value (day 0) of the 37°C group. Also after 4 or 5 days of culture at 37°C there is no significant difference in growth between the two groups. Fig. 2.4B shows that mineralization is arrested for 24 hours. The metatarsals started to calcify in the first 24 hours at 37°C, whether the culture started directly at 37°C or after a 24 hour preculture period at 22°C. Also, the length of the mineralized diaphyses of the two groups, when measured after 4 or 5 days of culture at 37°C, is not significantly different.

No differences were found between a culture temperature of 36°C (Biorack incubator C) and the standard 37°C (data not shown).

2.3.3 Effect of Na-b-glycerophosphate

Fig. 2.5 shows the effect of the addition of various concentrations of Na-b-glycerophosphate (bGP) to the culture medium. The length of the mineralized diaphyses of 16 day old rudiments (cultured in multiwells) was measured after a culture period of 5 days. It is clear that without bGP no calcification occurred. The maximum effect of bGP is reached at a concentration of 1.0 mM. Increasing the concentration up to 2.0 mM did not change the effect on mineralization.

Fig. 2.4. Effect of growth: total length (A) and mineralization: length of calcified zone (B) of 16 day old metatarsals directly cultured at 37°C (filled square) or with a 24 hour lag period of 22°C (open triangle). Media supplemented with 2.0 mM of bGP. Values are mean ± SEM, n=7.

2.3.4 Effect of Gas Phase

In all experiments a physiological sodiumbicarbonate buffer is used, which is the most common type of buffer applied in tissue culture. The use of this buffer requires a special gas mixture of air/5% CO₂. This poses some additional difficulties in the hardware development for Biorack experiments. Other buffers which do not require a special gas mixture are available but results from pilot experiments (data not shown) with HEPES as buffer (which requires only air as gas phase) show abnormal growth and mineralization, especially under CC. In the former experiments culture bags are placed in a 5% CO₂-incubator. However, in the Biorack experiment the culture bags will be placed in a closed Type-I containers. The remaining air volume in a filled container is relatively small and has to be flushed with the air/5% CO₂ gas mixture. We have compared the growth and mineralization of long bone rudiments cultured in polyethylene bags placed in an incubator or in bags mounted in a Biorack Type-I container. In a container the maximum number of 16 culture bags were placed. For the 4 day culture period containers were flushed at the beginning of the experiment and after 2 days (Fig. 2.6b), or only at the beginning of the culture (Fig. 2.6c). Long bones cultured in a standard multiwell placed in a CO₂-incubator were used as controls (Fig. 2.6a).

Both the increase in total length and the length of the mineralized diaphysis are less in containers closed for 4 days, compared to metatarsals cultured in the incubator. Cultures flushed after 2 days did not differ from the control cultures both in increase in total length and mineralization when measured at the end of the 4 day culture period.

Fig. 2.5. Effect of Na-b-glycerophosphate on the mineralization (expressed in length of calcified zone) of 16 day old mouse long bone rudiments cultured for 5 days. Values are mean ± SEM, *p £ 0.05 compared to 1.0 mM bGP, n=7.

We have adopted the use of double layered polyethylene culture bags for this experiment.^(14,18) When the medium is preconditioned with 5% CO₂ in an incubator, filling the bags with medium, ampoule, long bone rudiment and the subsequent sealing of the bag does not change the pH of the medium seriously.

When we consider growth (total length) of the rudiments it is clear from experiments shown in Table 2.I that cultures of long bone rudiments in polyethylene bags are very good comparable with cultures in conventional multiwells. Also, mineralization in culture bags (placed in a 5% CO₂-incubator) and in multiwells did not differ significantly. Applying continuous compression as a model for mechanical stimulation resulted in an increase in mineralization both in polyethylene bags and in standard multiwells (Table 2.I).

Also 17 day old fetal mouse long bone rudiments respond to mechanical forces.^(3,9) 17 Day old metatarsals have the same growth characteristics compared to 16 day old. Therefore we assume that 17 day old bones can be used as a resorption model in the same hardware.

We used continuous compression instead of intermittent compression, although the latter represents a more physiological condition and yields a higher response.⁽¹⁰⁾ However, to accommodate a pulsating device inside the relatively small Type-I container would leave very little free space and thus reducing the number of samples.

In the time line of microgravity experiments there is always a lag period between the handover of the experiment to the launch authorities and the activation of the experiment in orbit. Fortunately in Spacelab missions aboard the Space Shuttle this lag period is rather short. Latest handover is about 14 hours before launch, while Biorack is operational in about 10 hours after launch. Under tissue culture conditions there is the possibility to reduce biological activity during this lag period by reducing the amount of serum in the culture medium. In our experimental set-up this would mean that serum has to be included into the glass ampoule. Breaking of the glass ampoule should introduce serum into the medium and activate cell metabolism. However, tests to prepare glass ampoules containing serum showed that serum interfered unpredictable with the sealing procedure causing frequent leakages. Therefore we have included serum into the culture medium; the ampoules contain only the radioactive labels or fixatives. Lowering the temperature to 22°C, which is the ambient temperature of the Shuttle middeck stowage compartment, arrests the biological activity of the long bone rudiments during this lag period (Fig. 2.4). A culture period of 4 days (at 37°C), whether or not preceded by a 24 hour period of 22°C, resulted in the same increase in both the total length of the rudiment (Fig. 2.4A) and mineralization (Fig. 2.4B). Thus beginning with mounting of the culture bags in the containers, during handover, experiment integration and stowage, the containers must be kept at ambient temperatures (22°C). After activation of Biorack (in orbit) the containers are placed by the astronauts in the 36°C incubator where the tissues metabolism reaches normal in vitro levels rapidly. By breaking the ampoule with radioactive labels the incorporation of label can start only when under microgravity conditions.

Fig. 2.6. The effect of flushing a Type-I container with air/5% CO₂ mixture on growth (increase in total length) or mineralization (length of calcified zone) of 16 day old mouse metatarsals cultured for 4 days. a: control cultures in normal tissue culture plates in a CO₂ incubator (n=5). b: culture in polyethylene bags inside a Type-I container flushed at day 0 and after 2 days with air/5% CO₂ (n=5). c: as b but flushed only at day 0 and kept closed for the 4 day culture period (n=6). Media are supplemented with 1.0 mM bGP. Values are mean ± SEM, *p £ 0.05.

Our data show that 16 day old mouse long bone rudiments calcify readily during a 4 day culture period, provided that Na-b-glycerophosphate (1.0-2.0 mM) is added to the medium.

In multiwells long bone rudiments adhere with fibroblastic outgrowth to the bottom of the well which minimizes curvation of the rudiment during growth. However in culture bags adherence does not occur. We noted that to prevent curvation during growth not less than 1.0 mM Na-b-glycerophosphate should be added. We have no explanation for this phenomenon.

In culturing long bones in polyethylene bags placed into a Type-I container only a limited amount of gas mixture is present (about 2.0-2.5 ml gas mixture per 0.670 ml medium). Experimental conditions comparable with cultures in standard multiwells in a CO₂ incubator are met, providing the gas mixture is changed after 2 days (Fig. 2.6).

Because of limited stowage place available for Biorack, only a relative small pressure bottle can be accommodated. Therefor a total culture period of 4 days is chosen.

At the end of the experiment the samples for histological evaluations will be fixed and stored in the on board cooler, samples used for labeling studies (⁴⁵Ca, ³²P incorporation) will be stored in the on board freezer (-10°C).

We conclude that polyethylene culture bags, in combination with the Na-bicarbonate buffered aMEM medium supplemented with 1.0 mM of bGP and flushing of the experiment containers after 2 days with an air/5% CO₂ gas mixture, are very suitable to culture 16 day old long bone rudiments in Biorack Type-I containers. Under these culture conditions also the effect of compression can be studied in modified Type-I/E containers which are pressurized using the gas mixture from the pressure bottle. With the hardware used pressures can be monitored very precisely; PROD ± 0.5 kPa; Biorack data-lines ± 1 Pa.

With two different aged fetal mouse long bones (16 and 17 day old) as in vitro models it should be possible to determine the effect of microgravity on growth, mineralization and resorption in skeletal tissues without the interference of systemic factors occurring in vivo.

Since the culture method is based on conventional tissue culture techniques using a bicarbonate buffered medium, this hardware might also be used for the study of other tissues, organs, and cells under microgravity conditions.

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This study is supported by grant MG-004 from the Space Research Organization of the Netherlands (SRON).