Fetal Tissues Tested for Microbial Sterility by Culture- and PCR-Based Methods Can be Safely Used in Clinics

Abstract

Cell preparations to be used in clinical practice must be free of infectious agents. Safety concerns are especially elevated upon the use of human fetal tissues, which are otherwise highly advantageous in cell therapy. We demonstrate that treating fetal samples with antibiotic, extensive washing, and homogenization prior to cryoconservation efficiently removes microbes in general. Screening a large collection by an automatic culture system showed that 89.2% fetal tissue samples were sterile, while contamination was detected in 10.8% samples. Liver and chorion were contaminated more than the brain, kidney, lung, and soft tissues. Broad-range PCR from the bacterial 16s rRNA gene was adopted as a confirmatory assay; however, the concordance between the culture-based and PCR assays was weak. Taxonomic identification was done for contaminated samples by bacteriological methods and sequencing 16s rRNA PCR products. The two approaches revealed different spectra of taxonomic groups sharing only Lactobacillus, the most frequently found genus. In addition, other representatives of vaginal microbiota were detected by culture-based identification, while PCR product sequencing has also revealed a subset of nosocomial microorganisms. Importantly, species known to cause sepsis were identified by both techniques, arguing for their indispensability and mutual complementarity. We suggest that most contaminations are taken up during collection of fetal material rather than originating from an in utero infection. In conclusion, a rigorous microbiological control by culture and PCR is a prerequisite for safe clinical use of fetal tissue suspensions.

Figure 1

Microbial contamination in fetal tissue samples assayed by fluorescence in situ. Fluorescence staining with Texas Red (A, B) and SYTO-9 (C–E). (A) Lactobacillus rhamnosus culture (positive control). (B) Processed fetal tissue. Rod-shaped bacterial cells (arrows in A and B) occur among human cells and cell clumps in rare samples. (C) “Entry” sample (transportation medium used for harvesting the fetal material at the operation site). (D) A rare contaminated sample. Note round-shaped bacterial cells with the brighter area in the center likely representing the DNA. (E) The magnified portion of (D) outlined by a dashed square to show an individual bacterial cell. (F) A typical noncontaminated sample. Scale bars: 10 μm.

Figure 2

Polymerase chain reaction (PCR) showing an efficient removal of bacterial contamination by the processing procedure. Real-time PCR against 16s rRNA regions conservative for the entire bacterial kingdom [targeted by the BactQuant (A) and Jordan (B) primers], and regions specific for Lactobacillus (C) and human DNA (D). Abscissa: PCR cycle. Ordinate: relative fluorescence units (RFU). ENTRY: transportation medium used for harvesting the fetal material at the operation site. Liver, brain, soft tissues: samples prepared from the entry material. Mock: a water sample passed through the DNA isolation procedure to serve as a negative control. Note the much earlier rise of 16s rRNA PCR kinetics for “entry” in comparison to the prepared samples witnessing a dramatic reduction of bacterial DNA content after processing. The experiment has been repeated more than three times.

Figure 3

BacT/ALERT automatic culture from fetal samples. (A) Percentage of culture-positive samples of fetal liver (n = 200), brain (n = 193), soft tissues (n = 205), kidney (n = 32), lung (n = 36), chorion (n = 215), spleen (n = 51), and all samples (n = 932). (B) Tissue samples were first grouped by fetuses they were obtained from. Only fetuses providing four to eight tissues were considered. Then fetuses were ranged by the percentage of contaminated tissues as shown under the abscissa. Number (No.) of fetuses per each range is at the ordinate. (C) Percentage of culture-positive samples by the week of gestation: 5 (n =110), 6 (n = 111), 7 (n = 184), 8 (n = 241), 9 (n = 184), 10 (n = 45), 11 (n = 33), 12 (n = 30), and all samples with an identifiable age (n = 938). (D) The day at which the microbial growth was detected in BacT/ALERT culture bottles containing samples from the liver (mean = 1.34, n = 62), brain (mean = 1.57, n = 16), soft tissues (mean = 1.08, n = 25), and chorion (mean = 1.26, n = 59). The mean is indicated by the dash.

Figure 4

Detection of bacterial DNA in processed fetal preparations by PCR. PCR against 16s rRNA regions conservative for the entire bacterial kingdom [targeted by the BactQuant (A) and Jordan (B) primers]. Abscissa: PCR cycle. Ordinate: relative fluorescence units (RFU). Gray curves: dilutions of Escherichia coli (E. coli) DNA were used as standards. 1, 2, 3, 4: log genome equivalents per reaction. Dark curves: PCR from clinical samples labeled as “contaminated” and “clean” following the criteria described in the text. Mock: a water sample passed through the DNA isolation procedure to serve as a negative control. Insets: gel electrophoresis with BactQuant (447 bp) and Jordan PCR (371 bp) products from mock and clinical samples. Molecular weight is given in kilobases (kbp).

Figure 5

Microbial identification in fetal samples. (A) BacT/ALERT bottle culture was analyzed by bacteriological techniques. (B) Sequencing of PCR product from 16s rRNA gene (Fig. 4B, inset) followed by BLAST nucleotide database search. Dark portions of histograms correspond to the number (No.) of samples containing microorganisms known to pose a high risk of sepsis. White portions represent the samples bearing low-risk microorganisms only. Taxonomic grouping was done unevenly for a better representation of clinical importance.