A simple perfusion technique for isolation of maternal intervillous blood mononuclear cells from human placentae

Abstract

A noninvasive perfusion method for the recovery of maternal placental (intervillous) blood for use in immunologic assays is described. 60% of the perfused blood samples tested for fetal red blood cell (RBC) contamination were found to be pure maternal blood; in the remainder, fetal RBC contamination, with a single exception, was less than 6%. The intervillous mononuclear cells (IVBMC) isolated from this blood were of predominantly maternal origin as demonstrated by a polymerase chain reaction-based DNA typing technique. The number of IVBMC obtained was within the range of 9 to 55×10⁶ cells. Phenotypic analysis of IVBMC surface antigens revealed that 61% of the cells were CD3+ T-cells and 18% were CD19+ B-cells. The CD4+ and CD8+ T-lymphocyte subsets accounted for 28 and 26% of the IVBMC, respectively. The IVBMC were functionally competent as evidenced by in vitro lymphoproliferation and cytokine production in response to mitogen and PPD stimulation. This technique allows for rapid and safe isolation of large numbers of IVBMC which are functionally active up to 12 h post-delivery, thus representing a significant improvement over previously described methods. It should facilitate more vigorous research in the study of uteroplacental immunity and infectious disease research, particularly in field settings where sample collection and laboratory facilities are distant.

Introduction

The human placenta is a complex organ with several important yet disparate roles to play during gestation: it is responsible for maternal–fetal nutrient and gas exchange while at the same time acting as a barrier both against infectious organisms and maternal immunoreactivity to the fetus. Immunosuppressive soluble factors produced by the fetus, the placenta and the uterus (reviewed in Pope, 1990) have been strongly implicated in controlling maternal immune responses against fetal antigens and the role of the placenta as a physical barrier between the maternal circulation (and any infectious agents it may harbor) and the fetal blood has been well described. However, why this barrier sometimes breaks down, allowing infection of the placental tissue or fetus and how immunologic factors may influence these processes remains to be fully characterized.

Understanding maternal immunologic processes at the level of the placenta, particularly in the context of infectious diseases threatening fetal well-being, requires examination of intervillous blood and the cell populations within it. Methods previously reported for obtaining intervillous blood are problematic for a number of reasons. One of the simpler techniques that investigators have used involves cutting into the maternal surface of the placenta and collecting the blood that wells out of the incision. This method is suboptimal, however, since the placental blood, being rich in clotting factors, quickly coagulates, making collection of more than a few millilitres difficult. Another major disadvantage of this procedure is that the incision can damage the chorionic villi lying just beneath the maternal surface, resulting in contamination of the maternal blood with fetal blood. This is an important consideration in light of the reported immunosuppression cord blood lymphocytes exert over maternal lymphocytes (Papadogiannakis et al., 1984, Papadogiannakis et al., 1985; Shohat et al., 1986). Furthermore, accurate measurement of soluble factors such as cytokines or immunoglobulins in the intervillous blood is not possible when maternal and fetal blood are mixed. Finally, in light of human immunodeficiency virus (HIV) and other blood-borne infectious agents, use of sharp instruments in any blood or tissue-related methodology should be minimized in order to avoid the risk of accidental exposure.

Recently, Camelo et al. (1995)described the use of a sharp stylet to puncture the chorionic plate and thus gain access to the maternal blood in the intervillous space. They reported that >80% of samples collected in this manner contained less than 2% of foetal RBC contamination, but over 30% had extensive haemolysis. Also, while the small amount of blood obtained in this manner may be suitable for nutritional and physiological studies requiring small amounts of plasma, it is unlikely to yield sufficient material for cellular immunologic studies. This method is further limited in that it requires placentae less than 1 h old. Other workers have crushed or minced the placental tissue as a means of isolating blood; like the incision method, this procedure is unsuitable for obtaining blood intended for immunologic assays since maternal and fetal blood are freely mixed. Enzymatic digestion of placental tissue to release decidual immune cells has also been used; while simple and non-labor intensive, this procedure can lead to alteration of cell surface antigens (Ritson and Bulmer, 1987). A mechanical dispersion technique, in which placental tissue was teased apart to allow release of intervillous blood, was recently reported (Rasheed et al., 1992). The resultant mononuclear cells were functionally active, but this technique was characterized by a marked reduction in mononuclear cell viability when processing was delayed for more than 15 min following delivery. Furthermore, it is not clear to what extent this method allowed for mixing of maternal and fetal blood. The authors attempted to address the problem of contamination with fetal cells by testing for the presence of trophoblast- and amniotic epithelium-derived cells; thus, the extent to which fetal leukocytes contaminated the mononuclear cell preparations remains unknown.

Development of methodologies for the isolation of pure intervillous blood mononuclear cells (IVBMC) is essential to fully explore immunity and pathogenesis at the placental level, particularly in the context of infectious diseases in which the growth and development of the fetus are endangered. Many bacteria, viruses and protozoa have been detected in placental tissue and blood and can cross into fetal circulation, with outcomes ranging from preterm labor, low birth weight or intrauterine growth retardation to fetal abnormalities, spontaneous abortion, stillbirth or neonatal/post-neonatal death (Brabin and Brabin, 1992; Johnstone, 1992; Stray-Pederson, 1993; Johnson, 1994). An understanding of immune responses to infectious agents at the level of the placenta may provide insight into how such infections might be controlled or prevented and additionally may elucidate how poorly regulated immune responses can lead to placental immunopathology and compromised fetal outcome.

We report here the use of a perfusion technique, long used by students of placental physiology, to isolate pure maternal placental blood, which yields large quantities of functionally competent mononuclear cells. Application of this method to both laboratory and field locations should allow for more extensive characterization of the placental immune environment in the context of both normal gestation and during infectious disease processes.