In this day and age, mycoplasma contamination in cell culture laboratories is still a significant issue. We have dedicated a couple of blog entries to the issue of mycoplasma detection, yet important studies pointing to the severity of the situation are continuing to appear.
Recent statistical data points to contamination by Mycoplasmas as being an issue of realistic concern: A recent study by John Hogenesch’ lab at University of Pennsylvania Medical School analyzed 9395 rodent and primate samples from 884 projects in the NCBI Sequence Read Archive and found that 11% were contaminated by Mycoplasma. The authors further pointed out the fact that because these studies collectively totalled $3bn, it is likely that hundreds of millions of dollars in research funding are potentially affected, and likely negatively compromised.
This is a real concern. Elsewhere, BioData Mining published another study, wherein database corruption was investigated by analyzing sequences published in the 1000 Genome Project. The author, William Langdon at University College London, found that, when using Bowtie to map sequences, at least 7% were contaminated, in that they matched Mycoplasma but no human sequences.
Studies such as these highlight the real cost of Mycoplasma contamination when not taken into account.
Detecting contaminants such as Mycoplasma may not be easy. Low levels of contaminants may be hard to detect during initial screening, but later become visible during the bioproduction process, when it may be too late or when significant investment has already been made.
In our previous entires, we highlighted the use of new approaches for Mycoplasma detection based on Real-Time PCR as being more sensitive, rapid and efficient at detecting low level contaminants than the methods traditionally used.
These methods should become routine in lab use so that further development efforts are not met with products that have not been accounted for as being contaminated.
Akron Biotech is at the forefront of such efforts, and our Real-Time PCR Mycoplasma Detection Kit, Mycosolutions RT-PCR, has just launched as is now available for order.
Last week, we presented the first in our series of free educational webinars, titled “3D Nanofiber Scaffolds for Regenerative Medicine”. The webinar was aimed at both beginners to the field as well as advanced researchers by acting as both a primer on the current manufacturing and assembly techniques and applications of nanofiber scaffolds in biomedicine as well as an overview of the current state-of-the-art research and development in the field which touched on applications in cell culture, tissue regeneration, DNA transfection and drug delivery and transport, among others.
Questions posed by attendees during the webinar ranged from physical characterization of the electrospun nanofiber scaffolds, to the degree of manipulation of the assembly techniques to generate scaffodls of various mechanical properties to application-specific questions.
We want to thank everyone who attended the webinar: the interest and attendance was overwhelming, which shows the broad interest in the topic.
Due to high interest, we will be hosting the webinar again – the next date is October 23rd at 11 am ET.
To attend, use the registration link on this page:
We invite everyone interested in the subject the register and attend, and those who have attended our previous webinar should feel free to submit any questions or comments via email.
Also, if you have particular topics, questions or problems that you would like the webinar to address, feel free to get in touch with us via email.
We have also received a large number of requests for slides from the webinar: after October 24th, the webinar will be made available online. Check back here for details following the date.
Akron is inviting you to join us for the first in our series of free Regenerative Medicine and Tissue Engineering webinars. The series highlights some of the most interesting topics of relevance to professionals and anyone interested in new research, topics, processes and techniques in the Regenerative Medicine field.
The Webinar, titled “3D Nanofibers Scaffolds for Regenerative Medicine” will take place on Thursday, October 9, 2014 at 11 AM Eastern Standard Time (8 AM Pacific / 4 PM GMT).
About the Webinar:
The use of nanofiber scaffolds has emerged as a promising approach for tissue engineering and regeneration owing to the favorable chemical and physical characteristics of nanofibers which mimic the in vivo cellular environment. This webinar will review the current state-of-the-art of nanofiber scaffolds from their fabrication to their applications in tissue engineering.
The Webinar is free to attend and we invite everyone to participate. To register, follow this link:
We look forward to having you join us!
It has been almost a year since we first wrote about the importance of controlling your cultures against the onset of Mycoplasma contamination, and the statements we made back then are just as – if not more – important right now. The statistics – that 25% of all cultures are contaminated with Mycoplasma, and the limitations – that Mycoplasma is very hard to detect with regular light microscopes – still hold. Between then and now, the scientific community has increasingly highlighted the importance of developing and validating new methods for the more efficient detection of Mycoplasma in cultures.
The European Pharmacopiea 2.6.7 Mycoplasmas specifies the requirements for the validation of a nucleic acid-based test for Mycoplasmas. Because the potential consequences of Mycoplasma-contaminated cultures are significant, implementing rapid methods for the testing of cell therapy products can accelerate their validation and release, which is of significant therapeutic and commercial benefit.
With this in mind, the European Medicines Agency (EMA) approved a real time polymerase chain reaction (Real-Time PCR) Mycoplasma test in June 2013 for the testing and release of a lot of matrix-applied characterized autologous cultured chondrocytes (MACI®) which were approved back in April of the same year.
Mycoplasma RT-PCR detection systems are becoming more commonplace as an increasing number of scientific papers reports on their efficacy and speed. Regulatory agencies and validation programs are beginning to see the benefits that these detection systems bring and will begin incorporating them more thoroughly as part of release tests for cell therapy programs.
On 9 December 2014, the European Compliance Academy is hosting a Mycoplasma Testing Conference in Heidelberg, Germany which will feature many prominent speakers and companies. The one-day meeting will highlight new technologies for the detection of Mycoplasmas and the current research in the area, where RT-PCR systems are expected to take a central role.
With the above in mind, it is clear that containing Mycoplasma contamination is a critical aspect of cell therapy product development, and the awareness of the need for improved, more rapid detection approaches that will streamline the release process for cell therapies. We are curious to hear about your experiences with Mycoplasma contamination: do you think about it and how big of a part of your R&D/cell therapy process development scheme does it occupy? Write us or share your thoughts in the comments.
Finally, don’t forget to check out Akron’s own, industry-leading Mycosolutions RT-PCR kit, which will be available the first week of October.
Cord blood, as a source of progenitor cells, has been increasingly used in transplant procedures. However, transplant centers typically use cord blood units that have been processed and obtain by collection centers located elsewhere. This means that efficient cryopreservation procedures have to be in place for therapeutic use that lead to minimal cell loss. The key steps in cord blood banking include:
- Planning and preparation for cryopreservation
- Thawing at the transplant center
Typically, most cord blood banks assess potency, viability and hematocrit content prior to cryopreservation. Thawing of cord blood units after cryopreservation is often a tricky procedure, and one that may lead to the biggest variability in preserved cell viability.
Because thawing, washing and transfusion procedures vary among different centers, there has been an increasing call from the scientific community involved in this area for the standardization of these procedures across the industry.
A recent review paper published last month in Transfusion by Jeffrey McCullough and colleagues at the University of Minnesota reviewed current thawing practices performed across cord blood banks and identified inconsistencies among procedures before recommending elements to consider for more standardized thaw procedures across clinics.
Recommendations include decreasing thaw procedures and requiring validation of processing procedures at transplant labs as well as defining standards for processing. While comprehensive standards are currently not in place, there is an increasing need to provide more standardized approaches for the thawing of cryopreserved material. These recommendations are in addition to the standards for processing that organizations like FACT and AABB have have successfully put in place.
Colleagues in the field are echoing these recommendations set forth by Jeffrey McCullough’s manuscript. AABB and FACT have been called on to enhance inspection related to thawing/washing and transfusion and provide training related to these procedures. FACT has released guidelines with relation to administration of cord blood units. Similarly, AABB has been developing standards for blood banks and transfusion services since 1957.
For instance, AABB publishes Standards for Cellular Therapy Product Services which have formed the basis of AABB’s accreditation programs. Similarly, FACT-JACIE has published International Standards for Cord Blood Collection, Banking, and Release for Administration which can be found here. These, in addition to further initiatives aimed at providing guidelines to the industry, are laudable efforts on the part of FACT and AABB to achieve more comprehensive standardization across the industry. Numerous cord blood banks have been accredited for complying with FACT and AABB’s stringent cord blood banking guidelines.
The new recommendation is to apply the same efforts at providing standards for thawing, washing and transplantation.
New procedures that form the basis of the collective pool of scientific data are consistently appearing in the literature: just recently, a research paper was published outlining an improved procedure for the preservation of progenitor cells in preserved cord blood by storage at 4C prior to cryopreservation.
Whether such procedures can form part of cryopreservation approaches is part of the complexity of the assessment, validation and standardization procedure, and part of the reason why efforts in coming up with validated processes and unified standards have been challenging.
We are curious to hear about your experiences and thoughts about the issue of standardization of washing and thawing procedures for therapy. This is a subject that very much relies on user input, and the summary that we presented above all but scrapes the surface of the complexities of this problem. Nonetheless, we thought it worthwhile to draw attention to this issue so that we can begin this collective discussion. We welcome your thoughts as a comment on this blog or via email to email@example.com.
The RIKEN Center for Developmental Biology in Kobe, Japan, has had a tough year. Lauded at the start of the year for their discovery of STAP cells, the Institute later became the subject of controversy after uncertainty surrounding the papers resulted in their retraction and the suicide of one of the paper’s authors.
But now, hopeful news of a scientific move forward is coming from RIKEN. The Institute just started the first human trial using induced pluripotent stem cells, led by ophthalmologist Dr. Masayo Takahashi.
The media is quickly picking up this story, which involves using iPSCs for the treatment of age-related macular degeneration. Using iPSCs avoids the potential pitfalls associated with using embrokyonic stem cells in humans, owing to their improved immunoresponse and the absence of ethical issues.
Back in January, in an interview with New Scientist, Dr. Masayo Takahashi expressed confident optimism about the trials, but also explained that one of the disadvantages of her treatment – the extremely high cost – is because of the cells being derived from the same patient, as opposed to being allogeneic.
After the final safety green light, the first trial was performed on September 12th.
The patient was a Japanese woman in her 70s. According to RIKEN, a 1.3 x 3 mm sheet of retinal pigment epitelium cells was engrafted into the subretinal space of her eye.
The preliminary nature of this study implies an inherent uncertainty about its outcome, as well as potential risks associated with the treatment, which the scientists involved in the study are fully aware of, as was the patient.
Despite Takahashi’s initial optimism, there is no certainty that the study will reveal a successful therapeutic outcome. Nonetheless, this is a tremendous first step that the scientific community has welcomed with cautious, but hopeful, optimism.
Hypoxia – a term referring to an environment of reduced, or inadequate, oxygen supply – is not new to stem cells. In adult tissues, mesenchymal stem cells (MSCs) reside in environments of varying oxygen concentration, which is frequently below that of ambient conditions. This phenomenon has been receiving increased attention for its potential implications in regenerative medicine. A 2013 review suggested hypoxic culture conditions(2–5% O2 concentration) as being a promising alternative to current conditions for expanding MSCs.
Hypoxia is believed to increase MSC-related bone-healing processes, but the exact mechanism is not yet known. Recently, a number of papers have appeared investigating this phenomenon by analyzing osteogenesis under hypoxic conditions.
The first, from the Armed Forces Biomedical Research Institute in France, analyzed bone-healing efficiency in mouse models by subjecting them to hind-limb unloading. The authors found that bone-repair improvement occurred likely as a result of an improvement of natural bone-healing processes owing to the hypoxic conditions during remodeling, rather than the mobilization of an increased number of MSCs.
Elsewhere, researchers from the University of California Davis investigated how culture conditions (1%, 5% and 21% oxygen) affect the osteogenesis process, and found that hypoxia together with serum deprivation improved osteogenic differentiation of MSCs. The condition of 1% FBS and 5%O2 showed the highest concentration of alkaline phosphatase, which was used to characterize osteogenic efficiency.
Both of these papers are part of an increasing body of work showing how conditions far beyond what is currently considered the “norm” may shed light on improved cellular processes leading to tissue repair.