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 Wisconsin 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 28 1 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 2 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 Figure 1 Hypothalamic pituitarygonadal axis showing negative feedback loop from the gonads Adapted from Morohashi 1997 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 3 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 transplanted cells against a host immune response as the capsule prevents infiltration by immune cells and antibodies For the purpose of treating hormonal disorders the technology aims to allow therapeutic tissue transplantation without the use of immunosuppressant drugs which can have serious side effects Encapsulation of living cells was first described in the early 1950s but only in the last decade have significant advances been made in the area of microencapsulation Uludag et al 2000 In particular microencapsulation is advantageous due to small diffusion distances for nutrients gases waste products and hormones to travel to and from the encapsulated cells Hydrogels are crosslinked hydrophilic polymers that have been used extensively in the field of microencapsulation While hydrogels provide a desirable immunoprotective barrier their mesh structure also makes them highly permeable to oxygen nutrients and other molecules a necessary and attractive quality for maintaining viable encapsulated cells Most importantly efficient transport of hormone s in the HPG axis must be maintained for functionality of the encapsulation system 4 Depending on the hydrogel polymer crosslinking can be achieved via chemical temperature or light methods Photocrosslinking involves converting the liquid polymer to a hydrogel by free radical addition using a photoinitiator which generates radicals following absorption of ultraviolet UV or visible light The reaction is fast controlled and can be carried out under ambient or physiological conditions Nguyen and West 2002 In the context of cell encapsulation there are obvious concerns about the potential cytotoxicity of the photocrosslinking process However limitations can be overcome using mild conditions such as a low light intensity short irradiation time and low photoinitiator concentrations in the presence of cells Nguyen and West 2002 Prior to crosslinking the polymer must often be modified to include two or more reactive groups Figure 2 outlines the basic principles of encapsulation using mouse Leydig cells and a photocrosslinkable polymer Cells are first isolated from an animal donor and may be expanded in vitro under standard culture