Replica-molded poly(dimethylsiloxane) cell culturing lids contribute to cell culture longevity Axel Blau*, Tanja Ugniwenko, and Christiane Ziegler University of Kaiserslautern, Dept. of Physics & Biophysics, Kaiserslautern, Germany * Corresponding author. E-mail address: [email protected]

We present a new lid design to seal MEA glass rings against medium evaporation. It is based on replica-molded poly(dimethylsiloxane) and helps increasing cell culture longevity by means of stabilizing osmolarity at defined gas exchange. With such lids, osmolarity can be stabilized on average to increases of less than 5 mosmol/kg/month in humidified incubators, and to less than 10 mosmol/kg/month in non-humidified incubators when ¼ to ½ of the medium is exchanged on a 3-5 day schedule. However, if medium is not exchanged, osmolarity can increase by a factor of about 10 with respect to the aforementioned values. Extreme testing in a drying cabinet at 74°C yielded evaporation stability to 8 µL/h compared to 10 µL/h for PTFE-foil lids and 200 µL/h for regular Petri dishes. Incorporating pigments or objects to the pre-mixed resin furthermore allows adding other functionality to the lids. The lids were extensively tested on neural cultures of embryonic chicken.

1 Introduction Osmolarity affects neuroregulatory mechanisms [1]. In a follow-up study to work by Steve Potter [2] we present a new lid design for increasing cell culture longevity by means of stabilizing osmolarity and gas exchange. Quite often, even in humidified incubators the longevity of a cell culture is limited by a slow increase in osmolarity over time due to the evaporation of water. Using a simple replica-molding strategy based on poly(dimethylsiloxane) (PDMS), self-sealing lids for small sized culturing vessels can easily be fabricated. PDMS is a very flexible, autoclavable, biocompatible, and almost indestructible material of excellent transparency. Its refractive index is n = 1.43.

2 Results Two types of highly gas-permeable cell culturing lids made from PDMS are presented (figure 1). In one type, the optical properties were modified by pigments (e.g. for dark studies). In general, gas permeability can be tuned by embedding polymer windows (e.g. polyester). Once a template is available, unlimited numbers of lids can be fabricated.

Fig. 1 Transparent and pigmented PDMS lid for standard Ø 24 mm MEA glass rings.

When ¼ to ½ of the culturing medium is exchanged on a regular basis (every 3-5 days), the presented lids allow stabilization of osmolarity in humidified incubators (hi) to increases of less than 5 mosmol/kg/month, and to less than 10 mosmol/kg/month in non-humidified incubators (nhi). In contrast, osmolarity rises by approx. 30 mosmol/kg/month (hi) and by 80 mosmol/kg/month (nhi), respectively, when no medium is exchanged. Under the same conditions, osmolarity in regular Petri dishes rises by approx. 210 mosmol/kg/month (hi). A summary of the worstcase scenario is depicted in figure 2.

ISBN 3-938345-02-0

218

5th Int. Meeting on Substrate-Integrated Microelectrodes, 2006

Osmolarity [mosmol/kg] .

Comparison of changes in osmolarity over time

400

Teflon lid 35 mm Petri dish PDMS lid 24 well

350

300

250 0

5

10

15

Days in humidified CO2 incubator

Fig. 2 Comparison of the increase in osmolarity for PDMS-lids, PTFE-foil lids as described by Potter et al., 24-well cell culturing containers, and standard polystyrene Petri dishes.

Extreme testing in a drying cabinet at 74°C yielded evaporation stability to 8 µL/h for PDMS lids compared to 10 µL/h for PTFE-foil lids and 200 µL/h for regular Petri dishes. The new lids were extensively tested on neural cultures of embryonic chicken, which survived for more than 9 months; in all cases the cause of death was contamination.

3 Methods A simple template can easily be fabricated from a combination of two stacked plastic rods in a hollow cylinder (not shown). An adjustable template design is depicted in figure 3. It gives a choice of vertical lid geometries and membrane thickness. Lids were fabricated using poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow Corning) with high gas permeability but limited vapor permeability and negligible permeability for liquids. The two-component resin requires curing for 1 hour at 60°C or 0.5 hours at 85°C.

Fig. 3 Left: Sketch of the 5-part molding template in Perspex after assembly. PDMS is poured into the remaining cavity and cured at 85°C within 30 minutes. Right: Explosion view of the template. Holes and arcs are screw ports. This design allows arbitrary adjustment of vertical lid geometries and membrane thickness.

4 Summary While PDMS lids and PTFE-foil lids cannot prevent evaporation of water, they significantly help in keeping osmolarity constant when compared to regular cell culturing containers. Gas exchange with the environment is not compromised. Easy fabrication, robustness, and self-sealing properties make PDMS lids a suitable alternative to their PTFE-foil counterparts. Acknowledgements Many thanks to Simone Riedel, Jan Aigner and Tobias Budenz for excellent lab support. We would also like to thank the EU for partial funding of these studies (IST-2000-26463 FP5 IST FET open). References [1] [2]

Toney, G.M., et al., Central osmotic regulation of sympathetic nerve activity. Acta Physiol Scand, 2003. 177(1): p. 43-55. Potter, S.M. and T.B. DeMarse, A new approach to neural cell culture for long-term studies. J Neurosci Methods, 2001. 110(1-2): p. 17-24

ISBN 3-938345-02-0

5th Int. Meeting on Substrate-Integrated Microelectrodes, 2006

219