Important culture variables include: 1) tissue processing time and handling methods, 2) explant size and orientation, 3) HIV-1 inoculum type, dose and timing, 4) differences in culture medium and conditions, 5) protocols for adding microbicides to the tissues, and 6) assay endpoints. This subject is currently being addressed by the Microbicide Quality Assurance Program (MQAP), sponsored by NICHD and NIAID. Results from a multi-site comparison of HIV infection and microbicide efficacy were recently reported [19
]. Virus stocks, medium and microbicide formulations were standardized, and HIV infection of cervix, rectum and tonsil explant models was compared. In addition, a “soft endpoint” method was introduced to optimize and standardize data reporting. These measures decreased assay variability, although substantial inter-donor and intra-assay variations were still evident. This study also demonstrated widely different HIV growth profiles within the different tissue explant models, with unstimulated cervical tissue displaying very modest viral growth (less than 20% of that observed with rectal tissue, and less than 0.3% of tonsil tissue).
Modified cervical explant models have been introduced. To increase infectivity, target cells in explants have been activated with mitogen prior to HIV infection [5
]; this is an interesting approach that could mimic certain aspects of infection/inflammation, but the tissues are >48 hours old when they undergo HIV infection, and may be in an advanced state of deterioration. Polarized epithelial models, where tissue edges are sealed with agarose or Ussing chamber gaskets, provide an opportunity to study HIV infection across an intact epithelial surface, but because the tissue deteriorates quickly, cell viability and tissue permeability need to be closely monitored [20
]. Some researchers have begun to use previously frozen cervical tissue for microbicide testing [21
]; tissue integrity and cell viability issues are even more critical in this model. Current methods to assess tissue permeability and cell viability in explant cultures are relatively insensitive and nonspecific; new approaches are needed to enable precise assessment of permeability and viability at the cellular level within the tissues.
In summary, the current human cervical explant model generally consists of hormonally manipulated, topical microbicide (Betadine)-pretreated ectocervical tissue. Endocervical tissue, which may be more relevant, is rarely used. Several important variables known to affect HIV infection dynamics such as genital infections and inflammation, prior genital surgery, hormonal status and potential use of ARV or immunosuppressive drugs are usually unknown and would be extremely difficult to standardize. Betadine treatment, tissue manipulation and oxygen starvation can induce inflammation and compromise tissue architecture. In addition, several important physiological variables are missing. For these reasons, the current human cervical explant model cannot provide clear answers to many questions concerning HIV transmission.
Several steps can be taken to identify and limit the variables that may affect the outcome and data interpretation of HIV studies that use cervical explants. A first step would be to partner with the Ob/Gyn and Pathology teams working on the surgical cases. They could provide essential information from medical records, pathological observations obtained from gross and microscopic inspection of the tissue, and potentially provide a vaginal swab prior to surgery for detection of bacterial vaginosis (clue cells) and infectious pathogens (PCR diagnostic tests), cervicitis/vaginitis (granulocytes), and recent intercourse (PSA test). Tissue processing and culture techniques should be further optimized and standardized, and experiments designed to include extensive controls for quality assurance, entailing more rigorous measures of epithelial permeability and cell viability. Semen, cervicovaginal secretions and flora could be included as variables in some studies to more closely approximate physiological conditions. But even with all of these measures there will be a high degree of variability due to the intrinsic complexity of the model, and data obtained from HIV research on human cervical tissue should be interpreted with caution.
The recent MQAP study suggests that the human cervical explant model can produce reasonably consistent results when used for microbicide testing [19
]. Still, much work is needed on the design and interpretation of microbicide efficacy studies involving explant tissues. Several microbicide compounds that blocked HIV infection in preclinical cervical explants tests [3
] have subsequently failed in clinical trials [22
]. The main advantages to using cervical explant tissues instead of cell lines for microbicide efficacy testing are: 1) physiologically relevant target cells are present, 2) effects on barrier function and innate immunity can be addressed, and 3) effects of factors in the genital tract environment on microbicide efficacy can be studied. Protocols should be designed to capitalize on these strengths.
New in vitro models of reconstructed endocervical and ectocervical tissue are currently being developed and may circumvent several of the problems encountered with cervical explants [23
], but these of course will have caveats of their own.