Establishment of major histocompatibility complex homozygous gnotobiotic miniature swine colony for xenotransplantation

Abstract

To overcome shortages of human donor organs for organ failure patients, we made a commitment to develop gnotobiotic miniature swine as an alternative organ donor source for xenotransplantation. For this, we have constructed an absolute barrier-sustained gnotobiotic facility. Pregnant sows of gnotobiotic miniature swine, were procured and germfree piglets were obtained by hysterectomy. These were maintained in germfree isolators for about 4 weeks, deprived of colostrum and were fed sterilized soybean milk. They were associated with di-flora, anaerobic Lactobacillus sp. and Streptococcus sp. After confirmation of successful associations, gnotobiotic piglets were transferred into the facility aseptically. The piglets are maintained on high-efficiency particle air-filtered air in and out; maintaining constant room air pressure of 33 ± 3 mmAq, and sterile water and diet. In 10 sessions of hysterectomy, 18 male and 32 female piglets were obtained of which piglets (M six, F eight) died within 5 days. Among live piglets, piglets (M eight, F 12) were confirmed to be germfree by microbiological monitoring. For research of xenotransplantation, one consistent experimental result was essential. Therefore, major histocompatibility complex class II which related innate immunity, homozygotic gnotobiotic miniature swine was developed. As a result, genotyping revealed 14 individuals to be homozygous for major histocompatibility complex class II (DRB, DQB) as 0301, three individuals were homozygous as 0201 and each of two were homozygous for DQB as 0701 and DRB as 0404, respectively. Genetic modifications and immunological research for ideal alternative organ sources are in progress.

Production and rearing of germ-free X-SCID pigs

“…The improved isolator allowed us to deliver piglets from a uterus within 5 min. The resuscitation rate of piglets delivered within 5 min was 93% (14/15) in this study, which was higher than that reported in a previous report (72%, 36/50) [ 8 ] and comparable to that of another report (90%, 1,267/1,410) [ 14 ]. Therefore, a maximum time of 5 min for piglet derivation can be set as a criterion for successful germ-free delivery.…”

“…The production of germ-free pigs was first reported in 1964 [ 13 ] and in subsequent studies [ 8 , 14 , 19 ]. Piglets were obtained from pregnant pigs by hysterotomy and/or hysterectomy.…”

“…Hysterectomy appears to be better than hysterotomy because the former has achieved the shorter surgical time (30 min versus 4 h) and the higher germfree rate (91% versus 77%) [ 14 ]. The resuscitation rate of piglets delivered by hysterectomy, however, shows a wide range among studies [ 8 , 14 ].…”

Heterogeneity of porcine bone marrow-derived dendritic cells induced by GM-CSF

“…Gnotobiotic miniature pigs were kept under absolute barrier contained facility at the Bio-organ Research Center of Konkuk University, Seoul, Republic of Korea [22]. Animal experiments were carried out based on the National Institutes of Health guidelines for the care and use of laboratory animals.…”

The Use of Minipig in Drug Discovery and Development

“…This may be partly due to the fact outlined above that there are large differences between different origins of monkeys, for example, relating to major histocompatibility locus genetics. The pig has shown to be of use as an animal model in developing immunosuppressive agents (McDaid et al 2015), and transgenic colonies with defined histocompatibility complex can be established (Hwang et al 2015). Based on available knowledge (Bode et al 2010; Foster et al 2010) and recently accumulated data, the minipig is a valid alternative nonrodent species for safety testing of immunomodulatory development candidates and the functional similarity and the available tools make it definitely superior to the dog (Kammueller and Rubic in press).…”