The field of regenerative medicines includes not only cell- and tissue-based therapies, but also gene therapies. Human albumin has been exploited within a number of areas of regenerative medicine, but its main use until now has been within stem cell therapies.
Regenerative medicines and related therapies have great potential for treatment of a broad range of illnesses, including liver disease, cancer, cardiovascular disease and multiple sclerosis.
Albumin has been used as an ingredient in cell culture media for some time, and its abilities to stimulate growth of many cell types are well established. It has typically been applied in the form of serum. However, recent investigation has shown that it is possible to remove serum from stem cell cultures, but that it is preferable to have the presence of albumin.
Recently, the effectiveness of albumin in cell culturing has led to its occasional use in cryopreservation or formulation buffers for stem cell therapies.
This article will offer an outline of the use of albumin in the culturing, cryopreservation and formulation of stem cells, as well as explaining considerations of both a practical and regulatory nature.
Albumin at a Glance
With a concentration of approximately 40 g/L, human albumin is the most abundant protein found in blood. It is a single chain protein, made up of 585 amino acids and is approximately 66.4 kDa in mass [1]. Its compacted, heart-shaped structure contains 17 internal disulfide bridges. It acts as a buffer or reservoir in blood for a number of smaller entities, including metals, fatty acids, toxins and hormones. The combination of its abundant presence in a large number of tissues and areas of the body, as well as the free flow of albumin between these areas, enables albumin to transport these smaller entities between zones of high and low concentration. Moreover, albumin also makes up around 75% of the oncotic pressure of blood, and the sole free cysteine of albumin (at position 34) constitutes the majority of the reducing equivalents found in blood. Each of these traits is useful in the application of albumin for stem cell therapies.
Albumin in Stem Cell Culture
To begin with, the albumin used in stem cell culture was gathered from crude human serum or fetal bovine serum. Over time, the requirement for better defined, clearly characterized and easily controlled media has led to increased use of media made up of individually added proteins.
Since serum is made up of many constituents, some of which are advantageous to the stem cells being targeted, this shift away from its use has not always resulted in improved stem cell growth. As such, it was found that when replacing serum, it was necessary to include albumin alongside a number of growth enhancers, so as to ensure acceptable performance.
Nevertheless, despite adding albumin, and testing out a broad range of growth aspects, there are cases where users struggle to achieve satisfactory growth without the use of serum. The unknown factor or factors causing this stunted growth are often referred to with the light-hearted nickname, ‘pixie dust’.
While there are a number of academic laboratories which are content to employ bovine serum albumin for stem cell culture, the vast majority of industry users prefer to use human albumin to cultivate human stem cells, so as to avoid any complications relating to cross-species media.
It should be noted, however, that human serum albumin produced from human plasma is not a pure protein. The specifications for human serum albumins frequently allow for up to 5% of non-albumin proteins to be present. In addition, as human serum albumin will be derived from a pool of donor plasma and that will be different for each batch and country of origin, the number and type of ligands associated with the albumin will be different between batches and source. As such, products derived from serum cannot be used within media which claims to be chemically defined.
As a result, to attain this further level of control and safety reassurance, recombinant human albumin must be used.
Although there are cases where serum-derived albumin provides better growth, often due to passing on some of the aforementioned ‘pixie dust’ to the media [2, 3], this is an uncontrollable benefit. With a well-defined and pure recombinant albumin product the same effect can be obtained by adding in additional excipients but now with the benefit of actually knowing what supports the cell growth, making system control much easier and more consistent. In its own right the increased purity of the recombinant albumin can also lead to a greater performance in the cell culture [4]. Performance of serum albumin between batches can also be variable, with manufacturers often having to test each batch for its performance due to differences between the originating plasma pools.
The precise function of albumin in stem cell culture is still unclear [5], but over time, a number of biological properties have been ascribed to albumin, including:
- Providing nutrients
- Being a scavenger of toxins and reactive oxygen species
- Acting as a transporter and reservoir/sink for metals and other advantageous molecular entities
- Acting as a pH buffer
- Coating surfaces to cut down on direct interaction and act as an ‘insulator’ in media (thanks to its inclination to spread evenly throughout solutions)
It is most likely that the true function of albumin is a combination of all of these. It is also probable that the particular stem cell culture and precise conditions are what determines which properties of albumin are engaged and how much they are needed.
These complex functions of albumin may explain why it frequently makes an ideal base protein to add to a medium, as well as why it has become so popular, both in stem cell culture, and biology as a wider field.
There are three main factors for consideration when choosing an albumin source for use in commercial stem cell culture:
- Technical requirements
- Cost
- Regulatory compliance
As outlined above, the albumin product employed should enable the effective growth of undifferentiated stem cells in a manner which is robust and replicable. There is no bias for any of the albumin sources when it comes to technical requirements, but sources which lead to great variability between batches are difficult to manage in a cGMP setting.
As such, it is usually considered preferable to have a slightly lower yield which is replicable, as opposed to a higher yield with great variation.
Generally an increase in regulatory acceptability is accompanied by an increase in cost, as seen in Figure 1. The cost of goods increases from serum derived animal albumin through to human serum and again from this to serum-derived human albumin, with recombinant human albumin representing the highest costs.
From a regulatory standpoint, there is a parallel increase, with serum-derived animal albumin being rejected within commercial settings, and recombinant sources of human albumin representing the most acceptable form for the regulators.
The recent entry of recombinant human albumin – an animal-free, cGMP raw material of excellent consistency and purity - into the field of stem cell cultivation has allowed for culture media to be better defined and controlled. As well as enabling greater cell growth reproducibility, these media are preferred by the regulatory authorities, since there is comprehensive quality data available for all constituents.
Figure 1. Considerations for choice of albumin
Albumin in Stem Cell Cryopreservation
Cryopreservation eliminates the requirement for a continuous process, allowing for flexibility, and is thus an important stage in commercial stem cell therapies [6]. In general, it is preferable to have at least one cryopreservation step in one of the two possible positions in the process leading to the stem cell therapy dose.
The first position is upstream, usually as near to the harvest or production of the stem cells as can be managed. The second position is as far downstream as can be managed, preferably after the generation of the stem cell therapy dose.
Since it allows for multiple batches to be produced either in parallel or in combination (depending on the nature of the therapy), the first cryopreservation step can allow for economy of scale. The second cryopreservation step enables great flexibility in both distribution and administration of the therapy. When looking to produce a commercially viable product, these are both greatly desired qualities.
It has been demonstrated that recombinant albumin is more advantageous than plasma-derived albumin for the survival of stem cells during cryopreservation. For cryopreservation, stem cells are typically taken out of their growth medium, which supports their biological needs in growth and put into a cryopreservation medium.
In general, this medium does not contain any small molecular nutrients, but does have albumin, some salts and buffers, to uphold pH and isotonic conditions, and a range of cryopreservation agents, including DMSO, etc.
On formulation of the stem cells, they are rapidly frozen. Once thawed, they are either quickly returned to a growth medium, which will support additional processing, or placed in a final formulation solution. This is dependent on whether additional processing is required or if they are ready for administration.
The benefit of using albumin in cryopreservation likely stems from a combination of its properties, including its capacity to coat surfaces, its buffering abilities and its ability to stabilize entities in solution. These are all functions which support the stem cell in enduring the change in media and transition between phases during the cryopreservation.
However, the purity and the source of albumin are also important factors. It has been discovered that the use of yeast-derived recombinant albumin in cryopreservation can stop mesenchymal stem cells from continuing into late state apoptosis, when compared to plasma-derived albumin.
This leads to greater post-thaw viability for stem cells cryopreserved with yeast-derived recombinant human albumin against those with human serum albumin or albumin from other recombinant sources.
The considerations touched on in Figure 1 are relevant once more when considering the albumin source for cryopreservation use. The key decider is still the technical requirements, but the cost of goods has a lower impact on the choice here, since the quantities required for cryopreservation are significantly lower than those required for cell culture.
Albumin in Stem Cell Formulation
In order to reproducibly deliver an effective therapy, the formulation used for stem cell products is an important consideration. Usually, stem cell therapies are formulated immediately following their production or their post-cryopreservation thawing.
Following formulation, it is necessary that a number of time-consuming release assays be carried out effectively ahead of administration to the patient. As such, the longer it is possible to maintain the stem cells in a stable state at this stage, the better the applicability and adaptability of the therapy.
A general guideline is that a single day of stability allows for onsite administration, two days of stability will enable distribution nationwide and three days of stability opens up the possibility for global use.
A stem cell formulation which is prepared in a controlled medium typically allows for an easier release of the therapy, since studies can place emphasis on the stem cell therapy itself, instead of having to be on the lookout for possible effects and impurities arising from the medium. Additionally, there should be less variation in the background of biological assays, thanks to the formulation’s controlled nature.
Nevertheless, as there is crossover between the stages of the process, using controlled media only in the production of the final formulation is not sufficient. If the aim is to produce a controlled final formulation, this must be considered early enough in the process to make certain that a sufficient number of dilutions and exchanges have occurred to alleviate any risk from uncontrolled constituents.
As such, any controlled substances, including recombinant human albumin, must be brought in far before the final formulation.
Practical Considerations for Using Albumin in Stem Cell Therapies
The various functions of albumin discussed above are likely to require varying quantities for effectiveness.
Generally, it is helpful to test out both a high and low concentration of albumin, to experiment with its different biological properties. What these high and low concentrations will consist of is dependent on factors linked to the cell, such as type, concentration and phases, as well as the production process stage.
It is usually wise to begin with the high concentration representing around 2-5% of albumin, with the low concentration being around one tenth of this concentration. If advantageous impacts are perceived, it will be possible to further optimize the albumin content by experimenting with concentrations.
Recombinant human albumin, as produced by Albumedix in a yeast host, comes in a ready-to-apply formulation, with the albumin at 10% or greater concentration. Since this albumin material is so highly concentrated, it is usually simple to implement an easy addition or dilution of the liquid albumin, both in formulation studies and later, in large scale production.
Literature References
- Fanali G., di Masi A., Trezza V., Marino M., Fasano M., Ascenzi P.; Human serum albumin: from bench to bedside. Mol Aspects Med (2012) 33 209-290
- Horvathy, DB., Simon M., Schwarz, CM., Masteling M., Vácz G., Hornyak, I., Lacza, Z.; Serum albumin as a local therapeutic agent in cell therapy and tissue engineering. Biofactor (2016) doi:10.1002/biof.1337.
- Garcia-Gonzalo, FR., Belmonte, JCI.; Albumin-Associated Lipids Regulate Human Embryonic Stem Cell Self-Renewal, PLos ONE (2008) 3 e1384.
- Morback, ED., Paczkowski M., Frericksson, JR., Kirsher, RL., Hoff, HS, Baumann, NA., Moyer, T., Matern, D.; Composition of protein supplement used for human embryo culture. J Assist Reprod Genet (2014) 31 1703-1711.
- Francis GL.; Albumin and mammalian cell culture: implications for biotechnology applications. Cryotechnology (2010) 62 1-16.
- Hunt, CJ.; Cryopreservation of Human Stem Cells for Clinical Application: A Review. Transfus Med Hemother (2011) 38 107-123.
About Albumedix Ltd.
Too many people battle with diseases that keep them from living a full life. Healthcare professionals work hard every day to provide these people with better therapies. Together with partners, Albumedix utilize its albumin-based drug enhancing products and technologies to enable the development of more effective treatments.
With more than 30 years of experience, we are proud to be recognized as the world leader in recombinant human albumin products and technologies.
As the highest quality recombinant human albumin products ever developed, Albumedix enables the effective formulation of otherwise hard-to-stabilize drugs, cell therapies, and vaccines.
Our albumin-based technologies offer new ways of optimizing drug dosing and enhancing therapeutic performance by increasing the half-life, payload capacity, and tissue specific delivery of active pharmaceutical agents. This results in simpler treatment regimens, better performance, and, ultimately, improved patient outcomes.
Albumedix is headquartered in Nottingham, UK, with both research and large-scale manufacturing facilities. We are all committed to improving patient quality of life and are just as passionate about albumin and albumin-enabled therapies today as we were when we started 30 years ago.
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