Microencapsulation of Cells BME 400 University of Wisconsin—Madison December 7, 2005 Team: Bryan Baxter, Communications Director Timothy Eng, BWIG Representative Joe Zechlinski, Team Leader April Zehm, BSAC Representative Client: Craig Atwood, Ph.D., Sivan Vadakkadath Meethal, Ph.D., and Miguel Gallego, B.S. Department of Medicine, UW—Madison and Geriatric Research, Education, and Clinical Center William S. Middleton VA Hospital, Madison, WI Advisor: Assistant Professor Kristyn Masters Department of Biomedical Engineering University of Wisconsin1 Table of Contents Abstract…………………………………………………………………………………….. 2 Introduction Hormone replacement……………………………………………………................ 3 Encapsulation of cells……………………………………………………………… 4 Problem statement…………………………………………………………………………. 6 Biocompatibility…………………………………………………………………… 6 Immunoprotection………………………………………………………………….. 6 Encapsulated cell necrosis…………………………………………………………. 7 Design specifications………………………………………………………………………. 7 Biomaterials for cell encapsulation………………………………………………………… 8 Alginate/poly-L-lysine/alginate (APA)……………………………………………. 8 Hyaluronic acid (HA)……………………………………………………………… 10 Polyethylene glycol (PEG)……………………………………………………….... 11 Past work…………………………………………………………………………………… 12 Current design PEG biomaterial……………………………………………………………………. 13 Crosslinking scheme……………………………………………………………….. 14 Mouse MA-10 Leydig tumor cell line……………………………………………... 15 Microfluidic capsule generation…………………………………………………… 17 Current experiments Crosslinking studies………………………………………………………………... 19 Viability assays…………………………………………………………………….. 19 Microfluidic operation and cell encapsulation…………………………………….. 20 Cost analysis……………………………………………………………………………….. 23 Future work………………………………………………………………………………… 23 Conclusion…………………………………………………………………………………. 25 References………………………………………………………………………………….. 26 Appendix: Product Design Specifications…………………………………………………. 282 Abstract A method of microencapsulating Leydig cells for the long-term time release of male reproductive hormones in vivo is desired. These cells are currently being studied for use in anti-aging therapy. The final design must avoid issues of biocompatibility, immune responses and hypoxia commonly associated with microcapsule implantation. Currently, the encapsulation of mouse Leydig cells with diacrylated polyethylene glycol (PEGDA) using a constructed microfluidic device is being pursued. Crosslinking is achieved using a photoinitiator and 365 nm ultraviolet light. The viability of encapsulated cells is being examined; future work entails microencapsulation optimization and diffusion studies to assess capsule function in vitro.3 Introduction Hormone replacement The hypothalamic-pituitary-gonadal (HPG) axis regulates the hormones needed for reproduction including testosterone, inhibin, activin and others. Normally, in the male, production of testosterone is stimulated by release of leutinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary (Figure 1). These hormones act on Leydig cells to release testosterone, and on Sertoli cells to release inhibin and activin. Negative feedback to the hypothalamus and anterior pituitary acts to keep LH and FSH levels within normal limits (Widmaier et al., 2004). Natural aging results in the dysregulation of the HPG axis, and thus the loss of reproductive ability. It is hypothesized that with aging, a decrease in the ability to release adequate levels of testosterone and inhibin causes overproduction of LH and FSH by the HPG axis, and that such unregulated activity may be a causative agent for the wide-ranging diseases of aging in humans (Bowen and Atwood, 2004). Currently, androgen replacement therapy, which attempts to restore normal testosterone levels in the male, is achieved via oral administration, injections, or skin patches (Machluf et al., 2003). Successful testosterone therapy maintains normal serum levels of testosterone and its derivatives, and has been shown to increase muscle strength, stabilize bone density, and restore secondary sexual characteristics. However, significant side effects, such as hypertension and bone density loss, can arise from long term use and unsteady dose administration (Machluf et al., Figure 1. Hypothalamic-pituitary- gonadal axis, showing negative feedback loop from the gonads (Adapted from Morohashi, 1997).4 2003). Another disadvantage of these therapies is the requirement of multiple treatments and consequently, multiple clinical visits. Encapsulation of cells An alternative method for restoring the HPG axis is transplanting cells. This study attempts to design a novel method of encapsulating Leydig cells, which will provide an in vivo mechanism for the release of testosterone while providing a physical barrier to the host’s immune system. Such assisted hormone regulation, through the use of functional cells, should restore the normal function of the HPG axis by decreasing LH and FSH production in response to normal hormone release. Cell encapsulation provides a physical barrier for
View Full Document