Cell Biology International 31 (2007) 991e995 www.elsevier.

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Microbiological contamination in stem cell cultures

 Luis Corte s, Carmen Cabrera, Ana Nieto, Angel Fernando Cobo*, Jose Concha
Stem Cell Bank of Andaluc a (Spanish Central Node), Hospital Universitario Virgen de las Nieves, Avda Fuerzas Armadas 2, 18014 Granada, Spain Received 23 December 2006; revised 24 January 2007; accepted 11 March 2007

Abstract Cell therapy and regenerative medicine are potentially two of the most exciting aspects of the novel therapeutic methods currently under development. However, these treatments present a number of important biosafety issues, like the possible transmission of microorganisms to the recipients. The most common potential form of contamination in these cell products is by bacteria (including Mycoplasma), yeast and fungi. In our study, 32 stem cell lines and feeder cell lines were analysed. There were 19 contaminated cell passages (12%). The main contaminants were gram positive cocci and Mycoplasma species, followed by gram negative rods and gram positive rods. The Mycoplasma contamination rate was 4%. Stem cell banks and other research centres aim to screen all processed stem cell lines for these microorganisms, and to assure that no contaminants are introduced in the banking procedures. It is a standard part of current good practice in stem cell banks to carry out routine microbiological controls of the stem cell lines and to work in a controlled environment to reduce the probability of contamination in the nal product. 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.
Keywords: Stem cell cultures; Microbiological control; Cell therapy; Good practices; Clean rooms

1. Introduction Clinical treatment with stem cells consists of the transplant of these cell products to patients by means of systemic infusion or local injection, which raise unprecedented opportunities for the treatment of many diseases and traumas. In Spain, Andalusia was the rst region to pass a law (Law 7/2003, on October 20th 2003) about the research regulation using human embryos, non-viable for in vitro fertilization techniques and which have been stored in liquid nitrogen for more than 5 years. Later, a Spanish Royal Decree (2132/ 2004), with the establishment of the requirements and procedures to seek the development of research projects with stem cells obtained using spare embryos, was published on October 29th 2004. Recently, the Law 14/2006, concerning assisted reproduction techniques, which deals with the legal order on

* Corresponding author. Tel.: 34 958 02 03 75; fax: 34 958 02 01 32. E-mail address: fernando.cobo.sspa@juntadeandalucia.es (F. Cobo).

health care and research, above all in regenerative medicine (e.g. stem cell research, somatic cell nuclear transfer) was passed on May 26th 2006. After the regulation of the research activities in our country, scientists can legally handle stem cell lines and carry out clinical trials with these cells. For these purposes, stem cell banks and other research centres must assure the quality and safety of these cells, and these objectives are particularly important in avoiding the transmission of infectious diseases to the recipients of these cell products. Quality assurance is important in all aspects of cell culture. Good practice in the laboratory and the frequent monitoring of the stem cell lines is vital for any research centre or cell bank that handles cell cultures, and should be mandatory if these cultures are intended for clinical use in cell therapy and regenerative medicine. Also, this fact is important in order to avoid the transmission of infections to those who do research with stem cell lines. In this sense, stem cell banks aim to screen all processed cell lines for serious human or animal pathogens and to insure

1065-6995/$ - see front matter 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2007.03.010

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F. Cobo et al. / Cell Biology International 31 (2007) 991e995 stem cell line or feeder cell was handled in a biological safety cabinet type IIA (Richmond and McKinney, 1995) and two cell lines were never handled at the same time in order to avoid cross-contamination. Moreover, the technicians who handled the cell products always wore adequate clothes consisting of disposable sterile suits, boots, gloves and complete respiratory enclosure. The technicians who were working with these cultures were certied by the Spanish National System of Certication in laboratory techniques, and were all skilled technicians in this eld of the biology. Moreover, they attended external training courses periodically and at the same time, an internal training program was run. Furthermore, in our centre we introduced an environmental monitoring program to operate within a well documented quality assurance system for the air particle control, microbiological air contamination and surface contamination (Cobo et al., 2006).

that no contaminants are introduced in the banking procedures. Any microbe contaminating the donated material or being introduced as an adventitious agent during the manufacturing process can potentially multiply during the processing and can also potentially present a serious hazard to the recipients. The most common potential forms of contamination in cell cultures are bacteria (including Mycoplasma), yeasts and fungi, and these can be readily assessed on a routine basis (Cobo et al., 2005). The sources of microbial culture contamination are different: cell contamination from origin, glassware or apparatus (including storage bottles and pipettes), culture media, people and airborne contamination. To avoid contamination, a formal microbiological control program should be established and introduced to diagnose the main contaminants in stem cell cultures in order to avoid the transmission to possible recipients. In our bank, we have three years of experience with such a control program. In January 2004, we introduced a microbiological control program of both stem cell lines and feeder cells. Briey, this program consists of the systematic study of bacteria (including Mycoplasma), fungi and yeast contamination in all cell lines obtained in our centre in GMP conditions (European Union, 2003). The cells were cultured in different culture media, both solid and uid (e.g. Trypticase soy agar, uid thioglycollate, PPLO agar and uid) in accordance with the Standard Protocols given by the European Pharmacopeia (European Pharmacopeia, 2004a,b). The results of the sterility tests were recorded in registered documents and saved in electronic format in a data base. After this period, we evaluated the data obtained and we present these results below.
2. Materials and methods 2.1. Cell lines and their origins
Cell lines from different origins were used for this study from 1st February 2004 to 30th May 2006. The data records in the aforementioned period, were reviewed retrospectively. Thirty-two cell lines were tested in this study. The origins were diverse: feeder human broblasts (2), feeder mouse broblasts (12), mouse embryonic stem cells (9), mobilized human bone marrow (2), human bone marrow (5) and human embryonic stem cells (2). The CCD-1112SK cell line is a human skin broblast cell line with ATCC accession number CRL-2429. This cell line came directly from ATCC to our laboratory in March 2005 (cell passage 4). The H-181 and H-293 are two human embryonic stem cell lines that were obtained from the Karolinska Institute (Stockholm, Sweden). These human embryonic stem cell lines came directly to our bank in November 2004, at cell passage 46 and 47 respectively. The rest of cell lines were derived from our own bank. We evaluated both human and mouse broblasts because these kinds of cells are usually used as feeders in the growth of stem cell lines, so the contaminated feeders could transmit microorganisms to the recipients. The characteristics of these stem cell lines are provided in Tables 1 and 2.

2.3. Samples
Samples were taken systematically for sterility tests whenever cell lines were received or obtained and upon cell line sub-culture. The tests were carried out in all the consecutive cultures. At least 5e10 ml of specimens were used for sterility tests; after trypsinisation, both supernatant and cells were harvested for microbiological testing. All the cell lines analysed were cultured in antimicrobial free media before sampling for microbiological testing.

2.4. Sterility tests

The detection of bacterial, fungal and yeast contamination was carried out by means of standard culture-based sterility testing, in accordance with the pharmacopeia standards (Pharmaceutical Inspection Co-operation Scheme, 2002; European Pharmacopeia, 2004a,b; US Food and Drugs Administration, 2006). Specimens were cultured on four different media groups which include blood agar (Oxoid Ltd, Hampshire, UK) at 35  C, both trypticase soy agar and rieux, Marcy-LEtoile, France) at 35  C, broth thioglycolate mebroth (BioMe rieux, Marcy-LEtoile, France) at 35  C and dextrose Sabouraud dium (BioMe agar (Oxoid Ltd, Hampshire, UK) at 30  C and 35  C. The incubation conditions are shown in Table 3. Both solid and uid media were checked daily to ensure early detection of contamination and to enable appropriate actions to be taken as soon as the rst signs of contamination became apparent. The uid media were observed daily for turbidity. Turbid cultures were Gram stained. The broth was subcultured to select agar plates on the basis of the microbial morphology observed in the Gram stain. Later, the microorganisms were identied (see below). Although some studies were performed to demonstrate the sensitivity and rapidity of automated microbial detection systems (Kielpinski et al., 2005), we used the standard culture-based sterility testing because, at the moment, this is the routine method used in our laboratory and these methods are within the pharmacopeia standards. On the other hand, Mycoplasma detection was carried out by means of both standard culture and polymerase chain reaction (PCR). With respect to standard culture, we used both PPLO agar and broth (Biocult, Madrid, Spain) at 37  C 10% CO2 (see Table 3). Mycoplasma detection by PCR method was carried out by the use of the VenorGeM kit (Minerva Biolabs, Berlin). This kit uses the PCR technology for the detection of Mycoplasma and Acholeplasma contamination in cell cultures. The primer set is specic to the highly conserved 16S rRNA coding region in the Mycoplasma genome. This allows for detection of M. orale, M. hyorhinis, M. arginini, M. fermentans, M. salivarium, M. hominis, usually encountered as contaminants in cell cultures, but also M. pneumoniae, Acholeplasma laidlawii, M. synoviae and Ureaplasma species.

2.2. Good manufacturing practices in stem cell cultures 2.5. Microbial identication
Our bank is the central node of the bank network of the Spanish Cell Line National Bank. This is an establishment where both clinical use and research is being carried out. For clinical purposes, specic areas called clean rooms are used; however, the research is being carried out in separate conventional laboratories. In our bank, there are two clean rooms, of approximately 45 m2 of surface each, which are classied as grade B (European Union, 2003). Each The microorganisms isolated during the routine microbiological monitoring, except Mycoplasma species, were initially characterised by their cell morphology and Gram stain. Later, these microorganisms were identied by means of both an automated system (VITEK 2 compact, Vitek Systems, St. Louis, MO) and a manual system (in several cases in which the automatic

F. Cobo et al. / Cell Biology International 31 (2007) 991e995 Table 1 Main contaminants and characteristics of the stem and feeder cell lines Name of cell line Cell line origin Number of cell passages analysed 13 23 Number of cell passages contaminated 1 4 (a,b,c,d) Microorganism(s) isolated

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05 K 2 CCD-1112SK (ATCC No. CRL-2429) MEF 05 Y 0 05 Y 1 05 Y 8 05 Y 10 05 Y 11 05 Y 12 05 Y 13 05 Y 14 05 Y 16 05 Y 17 05 Y 18 05 U 1 05 U 2 05 U 3 05 U 4 05 U 5 05 U 6 05 U 11 05 U 12 05 X 1 05 SA 3 05 SA 4 05 H 1 05 H 2 05 H 3 05 H 4 05 H 5 H 181 H 293

Human broblasts Human broblasts

Mouse broblasts Mouse broblasts Mouse broblasts Mouse broblasts Mouse broblasts Mouse broblasts Mouse broblasts Mouse broblasts Mouse broblasts Mouse broblasts Mouse broblasts Mouse broblasts Mouse embryonic stem cells Mouse embryonic stem cells Mouse embryonic stem cells Mouse embryonic stem cells Mouse embryonic stem cells Mouse embryonic stem cells Mouse embryonic stem cells Mouse embryonic stem cells Mouse embryonic stem cells Mobilized human bone marrow Mobilized human bone marrow Human bone marrow Human bone marrow Human bone marrow Human bone marrow Human bone marrow Human embryonic stem cells Human embryonic stem cells

27 4 4 1 2 1 1 1 1 1 1 2 2 1 25 2 3 1 9 7 3 1 2 2 6 1 3 4 2 2

3 (a,b,c) 0 0 0 0 1 1 1 1 1 1 1 0 0 1 0 0 0 0 1 1 0 0 0 1 0 0 0 0 0

S. hominis (a) S. hominis and Granulicatella adiacens, (b) Granulicatella adiacens and M. luteus, (c) S. epidermidis and S. warneri, (d) Corynebacterium spp. (a) Burkholderia cepacia, (b) Granulicatella elegans, (c) Corynebacterium spp.

Mycoplasma Mycoplasma Mycoplasma Mycoplasma Mycoplasma Mycoplasma Mycoplasma

spp. spp. spp. spp. spp. spp. spp.

S. hominis

Stenotrophomonas maltophilia Acinetobacter lwofi

Micrococcus luteus

identication was not possible). Moreover, the determination of susceptibility of strains was carried out using the same system. Positive internal quality assurance control was introduced in order to check that the culture procedures were correct. The growth promotion test for each batch of standard test media was carried out using reference strains specied by the pharmacopoeial method: Staphylococcus epidermidis (CECT 4183) for trypticase soy agar and uid, Staphylococcus aureus (ATCC 6538) for blood agar, Micrococcus luteus (CECT 244) for uid thioglycollate medium, Candida albicans (ATCC 10231) and Penicilium chrysogenum (NCTC 2802) for dextrose Sabouraud agar, Mycoplasma orale (NCTC 10112) and Mycoplasma pneumoniae (NCTC 10119) for PPLO agar and uid. Table 2 Number of cell passages vs. contaminated cell passages Cell line origin Human broblasts Mouse broblasts Mouse embryonic stem cells Mobilized human bone marrow Human bone marrow Human embryonic stem cells Total Number of cell passages 36 46 53 3 16 4 158 Contaminated cell passages 5 10 3 0 1 0 19

The procedures with the growth promotion test have been published in the European Pharmacopoeia (Pharmaceutical Inspection Co-operation Scheme, 2002). For this purpose, 10e100 colony-forming units of each microorganism were inoculated by duplicating the media. The inoculum was veried by viable plate counts. Positive controls were tested in the test media within 3 days for bacteria, 5 days for fungi and yeast and 21 days for Mycoplasma. Negative controls were carried out by means of inoculation of sterile water in the media. An equivalent number of negative control samples to the test samples were carried out. The temperature of incubators was also registered daily by reading the incubator LED. Moreover, a daily control of the incubators used for sterility tests was carried out by means of an external thermometer (e.g. Data Logger Thermometer CENTER 305, Center Technology Corporation, Taiwan).

3. Results In this study, thirty-two stem cell lines and feeder cell lines were analysed (Table 1). Overall, 158 cell passages were monitored by means of microbiological cultures using the methodology described in Section 2. There were 19 contaminated passages (12%). The main contaminants were gram positive cocci (10) and Mycoplasma species (7), followed by gram

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Table 3 Culture media and incubation conditions for the microorganisms isolated in stem cell cultures Microorganism Bacteria Trypticase Culture media Blood agar Soy agar Fluid thioglycollate medium Fluid trypticase soy PPLO agar PPLO uid Dextrose Sabouraud agar Dextrose Sabouraud agar Incubation temperature 35  C 35  C 35  C 35  C 37  C 37  C 30  C 35  C Incubation atmosphere Aerobic Aerobic Aerobic Aerobic 10% CO2 10% CO2 Aerobic Aerobic Days of incubation 14 14 14 14 14 14 14 14

Mycoplasma Fungi Yeasts

PPLO, pleuro-pneumonia like organisms.

negative rods (3) and gram positive rods (2). Three cell passages were contaminated by two microorganisms (see Tables 1 and 2). With respect to Mycoplasma testing, the culture of this bacteria was negative in all cases, but by means of the PCR method seven cell passages were positive. The Mycoplasma contamination rate was 4%. With respect to the positive control results, the reference strains mentioned above grew in all batches of the culture media used for standard culture-based sterility testing. On the other hand, all the media inoculated with sterile water were negative. 4. Discussion Cell line contamination is the main problem in research centres that work with cell cultures. There are four major sources of microbiological contamination: people, air, media and reagents and cell lines. The most important source of contamination is the contaminated cells that are used as the primary starting material for cell culture. The origin of the cell lines can have an important effect and the cell lines most recently imported can constitute the greatest source of contamination, depending on their culture history and past exposure to microorganisms. Thus, providers of cell lines should be able to provide details of cell passage history and appropriate testing (Stacey and Phillips, 1999; Stacey et al., 2000). So, when a new cell line enters the centre, it should be considered under quarantine status. Another important source of contamination, apart from cell lines, is the people who handle the cell cultures; so, the main contaminants will be the microorganisms present in the saprophytic human skin ora. People disperse hundreds and sometimes thousands of bacteria per minute into the air (Whyte and Bailey, 1985). Thus, the environment in which the cultures are carried out, will have thousands of microorganisms that can be deposited into the cultures. Other potential sources of microbial contamination in cell cultures include the media and reagents, the laboratory environment and the break-down in aseptic processing. In our study, we observed that the majority of the microorganisms recovered were opportunistic human pathogens, which are inhabitants of normal human skin, mucosal, oropharynx and also in the environment. These results are in accordance

with a paper we recently published about environmental contamination in clean rooms (Cobo and Concha, 2007). So, these microorganisms can travel through the air, deposit themselves into the cell cultures and later produce contamination. There are several recent reports of microbial contamination in cell cultures (Langdon, 2004; Mirjalili et al., 2005) and the most frequently isolated microorganisms are Mycoplasma, other bacteria from the human skin, airborne bacterial spores, airborne fungal spores and most infrequently gram negative bacteria (Halls, 2004; Owers et al., 2004). In our study, we observed that the majority of microorganisms recovered from stem cell cultures were saprophytic pathogens, which are inhabitants of normal human skin, mucosal, oropharynx and also in the environment. Moreover, Mycoplasma was recovered in seven cell cultures. Mycoplasma is still on top of the list of the major threats for cell cultures (Uphoff and Drexler, 2005). A recent report obtained a 19% contamination rate by Mycoplasma species, although, by incorporating the mixed contamination results in which Mycoplasma was the main isolate, led to the Mycoplasma contamination rate rising to 22% (Mirjalili et al., 2005). However, in the present study our results revealed an overall Mycoplasma contamination of 4%. So, our contamination rate is lower than that in previous reports. Moreover, we used two methods for the diagnosis of Mycoplasma species, because it is usually recommended to use at least two techniques for testing to ensure optimum sensitivity and specicity (European Pharmacopeia, 2004b). Furthermore, the percentage of overall contamination in our study was 12%; compared with other reports (Mirjalili et al., 2005) our overall contamination rate was thus lower than in these articles. Why was our contamination rate lower? We suspect that it was our working in the previously described clean rooms, wearing appropriate clothing and applying strict rules of good practice that could be the explanation of this fact. Furthermore, the application of microbial and environmental control programs in stem cell cultures could permit the use of antibiotic free cell cultures in order to avoid the harmful effect of antimicrobials in cell cultures (Doyle and Grifths, 1998) and the potential disguise of any potential source of contamination. Although the contamination of cell cultures in all types of laboratories is not recommendable for many reasons, this fact should be unacceptable in cell products for use in clinical trials

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or in cell therapy. Stem cell banks arose out of the necessity to provide a reliable and reproducible source of stem cell lines which includes the assurance of safe starting materials for use in human therapy that have also been checked for authenticity, stability and performance. Routine screening for stem cell lines helps in the early detection of contamination, since any type of manipulation is a potential source of contamination. In order to avoid the contamination, control and trend analysis of stem cell cultures and their possible contaminants is necessary. The isolation frequency and the microbial identication should be carried out to establish trends and to demonstrate sterility control. Finally, stem cell banks aim to screen all processed cell lines for both human and animal pathogens and insure that no contaminants are introduced in the banking procedures, including storage. Thus, apart from implementing routine good practices for the handling of stem cell cultures, the adequate preparation and introduction of a microbiological, environmental and biosafety quality assurance program is mandatory. Acknowledgements Ms Angela Barnie is acknowledged for the English correction of this manuscript. We also thank Dr Bernat Soria and Dra Outti Hovatta for the H-181 and H-293 human embryonic stem cell lines. References
Andalusian Law 7/2003, October 20th. Research regulation using human emn Ocial Junta bryos non-viable for in vitro fertilization techniques. Bolet a. de Andaluc Cobo F, Concha A. Environmental microbial contamination in a stem cell bank. Lett Appl Microbiol 2007;44:379e86. Cobo F, Stacey GN, Hunt C, Cabrera C, Nieto A, Montes R, et al. Microbiological control in stem cell banks: approaches to standardisation. Appl Microbiol Biotechnol 2005;68:456e66.  Environmental monitoring in stem s JL, Concha A. Cobo F, Stacey GN, Corte cell banks. Appl Microbiol Biotechnol 2006;70:651e62.

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