Making tissue culture commercially viable

The science and, indeed, art of tissue culture is almost 100 years old. As R. Ian Freshney states in the preface to the indispensable manual of tissue culture, Culture of Animal Cells: A Manual of Basic Technique, "Tissue culture has been in existence since the beginning of the last century and has passed through its simple exploratory phase, a later expansive phase in the 1950s, and is now in a specialisation phase focusing on control mechanisms and differentiated functions."

Tissue culture was originally developed as a technique for overcoming the obstacles that the body poses to scientific experimentation, and it is often described in terms that are diametrically opposed to any classical definition of the body: where the body is whole, tissue culture is fragmented; where the body is opaque, tissue culture is transparent; while the lifespan of any body is finite, cell lines can be 'immortalised'.

Techniques for culturing cells were not universally implemented until World War II, which proved the catalyst for widespread use. From the mid-1940s onwards, huge collections of tumour and orthopaedic tissue were built by organizations such as the American Tissue Culture Collection (ATCC) founded in 1947 and the US Navy Tissue Bank founded 2 years later. By the 1950s, tissue culture was becoming mainstream: in 1952 a report in Cancer Research described the first human tumour cell line, the 'HeLa' line derived from cervical carcinoma tissue. This line has gone on to be perhaps the most widely used line in the history of tissue culture.

Tissue culture is now being used to rebuild missing or damaged tissues; that is, to 'reconstruct' those tissues, as opposed it its original incarnation; a 'deconstructionist' approach where the understanding of an intact organ system is gleaned from understanding of the relevant cell biology.

Fibroblast propagation

Fibroblast propagation is an excellent example of one of the simplest approaches to tissue culture: a fragment of tissue is placed on a solid substratum where, following attachment, cell migration is encouraged. Once the cells have been established, they can be reproduced repeatedly. Cells are first allowed to grow to confluence (covering all of the available growth area in the vessel in which they are being cultured). This is an important point as 'normal' cells —those that have not been given a selective growth advantage, either by the nature of the tissue from which they were obtained (for example, tumour tissue) or by the investigator (usually by genetic intervention in normal cell growth control mechanisms) — become 'contact inhibited' and stop growing (Figure 1).

Figure 1 Fibroblast confluence chart.

Growing cells

The conditions in which this growth occurs are also well established and can be summarized as:

• the gas phase

• the liquid phase

• nutrient supply

• temperature.

Most eukaryotic cells, similar to the organisms they are derived from, require oxygen for normal function, which is delivered via atmospheric air. Although slightly counterintuitive, carbon dioxide also needs to be delivered to fulfil a role in maintenance of pH, normally at 5% with the balance being atmospheric air. This leads onto the liquid phase (culture media), which contains buffers for pH control (often bicarbonate based salts) around 7.4 (physiological pH); sometimes a pH indicator (phenol red that turns yellow in more acidic conditions and pink in more alkali conditions); additional salts to maintain osmolality (around the human plasma level of approximately 300 mOSm/kg); essential amino acids and vitamins; and an energy source, glucose, all dissolved in an H₂O base.

Definition

Tissue culture serum (bovine, equine, human) is then added. Most human cultures require serum because of the known and unknown compound it contains; it is believed that the majority of these are polypeptides, such as growth factors including PDGF, EGF, FGF, IGF and hormones, especially insulin.

It has long been a goal of tissue culture to remove the need for serum because of the confounding effect it has on any cellular observations that are made and to have more direct control over the cellular environment.

Many studies have now shown that serum can be reduced or replaced in the culture of many cells if the correct nutritional and hormonal adaptations are made to the basal, non-serum-containing medium. This is a time-consuming and laborious process (as all combinations are tested) often leading to a serum-free solution that does support growth, but not at the level seen when serum is included.

Key points

Finally, cells from homeotherm/endotherm (warm-blooded) organisms must be maintained at 'body' temperature, 37 °C. This culture media is changed when nutrients are used up, too many acidic waste products have been produced or when gas buffering is impaired; the presence of the phenol red indicator is a great help for this. With growing cells, this happens every 2–3 days.

When the cells have filled the vessel they require 'subculturing', that is removal from the growth area and 'passing' to an enlarged growth area to allow continued growth. It is often asked why cells are not grown in sports-field-sized vessels.

Apart from the obvious physical constraints, we also know that 'normal' cells need to be in reasonable proximity to each other (autocrine effects) to proliferate happily. Cell attachment is mediated by the presence of Ca²⁺ /Mg²⁺ ions, therefore, the removal of cells is made easier by the absence of these ions. A proteolytic enzyme, such as trypsin, is also delivered in such an environment, as this will 'dissolve' proteins holding cells to the substratum. This process removes the cells from the growth area so that they can then be redistributed amongst more growth areas. Consequently, the process continues.

EU regulations

Although primary cell culture techniques have remained relatively unchanged for many years, the ability to use human cells in medical applications to treat disease and injury is hampered by a variety of issues.

There are over 250 operations in the EU in various stages of development involving tissue engineering, regeneration and subsequent attempts at commercialization. The primary division in the emerging industry is the classification of autologous versus allogenic applications.

Autologous means that donor and patient are the same. Allogenic applications use one or few donors and reproduce for many potential patients. Ethical debates continue over the use of allogenic donors, but for the most part, the debates have centred on the use of human stem cells for the development of a variety of medical applications.

As the industry continues to develop, the commercialization challenges are apparent. The requirements of scalable and consistent manufacturing platforms are one of a number of barriers to market entry facing cell culture operations. Although it has been shown for many years that cells can be cultured successfully in a research laboratory, the requirements of a commercial laboratory are quite different.

The design, implementation and execution of quality standards similar to market expectations in the pharmaceutical industry is an absolute necessity. EU regulators are keeping a keen eye on the operating environments of this relatively new sector. New standards have been developed and will begin to take effect this year. Compliance with these standards will present a challenge to many fledgling cell culture operations.

Large-scale cell manufacturing

Beyond the regulatory environment, the manufacturing environment requires significant improvements. As primary cell culture is a labour-intensive operation, the emergence of automated cell culture platforms will accelerate successful market entry. The ability of autologous commercial operations to scale their operations is dependent on the use of automated systems; though there is an inherent variance in the growth rates of cell cultures. There needs to be major reductions in the costs associated with cell culture techniques, particularly in regard to autologous cell applications. If economies of scale are not achieved through scalable manufacturing systems, autologous operations will be restricted to niche applications.

Many medical professionals believe that the ability of the human body to repair and regenerate itself by utilizing its own cells for both organ and tissue repair is the future of many medical treatments. The size and scope of the potential market is enormous. This factor alone will make it possible for the continued development of systems to safely and effectively produce cell culture products.

The 72-hour race

However, manufacturing platforms and quality issues are not the only barriers facing many medical development firms in this industry.

Consider the logistical challenges that these companies face. For example, in autologous cell applications, the effective life span of the produced 'product' is approximately 72 hours post-cellular harvest. In other words, all of the production control tests, quality control (QC) tests, quality assurance (QA) tests and packaging requirements take place in the most time-sensitive environment.

Compared with a pharmaceutical firm manufacturing in batches comprising thousands of lots per batch, these firms are manufacturing in batch lots of one. The record keeping, coordination and synchronization between the cell provider and cell user is extraordinary.

Alongside the internal challenges these companies face, the external challenges are equally daunting. If the product you are manufacturing has a shelf life of 72 hours, the requirements of coordinating on time delivery to specific destinations in the EU within 24–48 hours is problematic.

Although delivery systems have improved substantially over the past few years, they are still not reliable enough to meet many medical requirements. This is particularly evident in areas outside the major metropolitan centres.

Compounding the logistical issues of delivery to medical treatment centres is the human interface. The reality is that physician and patient schedules often change without notice.

If either physician or patient has last minute changes to their schedule, the cell product may not be administered prior to cell quality expiration. Because many of these cell applications are administered on an outpatient basis, it is essential that both physician and patient understand the restrictions of these biological timelines in relation to cellular viability.

This time restriction also requires extraordinary synchronization of scheduling between the producer, the delivery system, the hospital staff, the physician and the patient. Aligning these stars consistently is another barrier to a scalable industry application.

Education

Those issues notwithstanding, there remain key areas that many firms face as they contemplate the progression from R&D to commercialization. Educating the consumer and physician communities in the safety and efficacy of cellular treatments is an integral component of the process.

The majority of physicians are not yet equipped to understand the complexity of cell culture, the physiological response of cells transplanted into the tissue or organ and the predictability of patient response.

What does the future hold? One would expect major improvements in the cell culture process. Additionally, we would expect cryopreservation techniques to be developed that extend the viability of cell productions and that will eliminate the majority of logistical challenges these firms face. Applications continue to expand and the endorsement of medical institutions to utilize these techniques will grow as well.

Bibliography

1. R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique 3rd Edition (Wiley-Liss, New York, NY, USA, 1994).

2. K.A. Lygoe and M.P. Lewis, A 150-Year History of Autologous Treatements in Western Medicine (2004).

3. A. Bayat, D.A. McGrouther and M.W.J. Ferguson, BMJ, 326(7380), 88–92 (2003).

4. E. Bell et al.,Science, 211(4486), 1052–1054 (1981).

5. M. Brittberg et al.,N. Engl. J. Med. 331, 889–895 (1994).

6. M. Brittberg et al.,J. Bone Joint Surg. Am. 85-A Suppl. 3, 109–115 (2003).

7. R.A.F. Clark, "Wound Repair: Overview and General Considerations," in R.A.F. Clark, Ed. The Molecular and Cellular Biology of Wound Repair (Plenum Press, New York, NY, USA, 1996) pp 3–50.

8. C.P. Denton and D.J. Abraham, Curr. Opin. Rheumatol. 13, 505–511 (2001).

9. C. Erggelet, M. Sittinger and A. Lahm, Arthroscopy: J. Arthrosc. Rel. Surg. 19(1), 108–110 (2003).

10. M.W.J. Ferguson, Br. Dent. J. 192(8), 475 (2002).

11. D.W. Hutmacher, J. Biomater. Sci., Polym. Ed. 12, 107–124 (2001).

12. R. Kato et al., Exp. Cell Res. 265, 54–63 (2001).

13. G. Keller et al., Bull. Exp. Biol. Med. 130, 786–789 (2000).

14. P.T Khaw et al., Curr. Opin. Ophthalmol. 12, 143–148 (2001).

15. R.S. Kirsner, V. Falanga and W.H. Eaglstein, Trends Biotechnol. 16(6), 246–249 (1998).

16. T.R. Knapp, E.N. Kaplan and J.R. Daniels, Plast. Reconstr. Surg. 60, 398–405 (1977).

17. M.P. Lewis and J.T. Norman, Exp. Nephrol. 6(2), 132–143 (1998).

18. T.S. Lin et al., Bone Marrow Transplant, 29, 763–767 (2002).

19. T.S. Lin et al., Curr. Hematol. Rep. 2(4), 310–315 (2003).

20. I.A. McGregor and A.D. McGregor, Fundamental Techniques of Plastic Surgery, (Churchill Livingstone, New York, NY, USA, 1995) pp 35–59.

21. W.W. Minuth, M. Sittinger and S. Kloth, Cell Tissue Res. 291(1), 1–11 (1998).

22. C.J. Mullany, Circulation 107, e21–e22 (2003).

23. J.S. Munger et al., Cell 96(3), 319–328 (1999).

24. A.N. Neely et al., Wound Repair Regen. 7(3), 166–171 (1999).

25. L. Peterson et al., Proc. Orthop. Res. Soc. 9, 218 (1984).

26. L. Peterson et al., J. Bone Joint Surg. Am. 85, 17–24 (2003).

27. T. Philip et al., N. Engl. J Med. 333, 1540–1545 (1995).

28. M.L. Sabolinski et al., "The Clincal Experience of Bioengineered Skin Products," in V. Falanga, Ed, Cutaneous Wound Healing, (Martin Dunitz Ltd, London, UK, 2001) pp 411–432.

28. D. Sheppard, Chest 120 Suppl. 49–53 (2001).

29. A.M. Sieuwerts et al., Int. J. Cancer. 76(6), 829–835 (1998).

30. J.J. Tomasek et al., Nat. Rev. Mol. Cell Biol. 3(5), 349–363 (2002).

31. J.A. Tuxhorn, G.E. Ayala and D.R. Rowley, J. Urol. 166, 2472–2483 (2001).

32. D. Watson et al., Arch. Facial. Plast. Surg. 1(3), 165–170 (1999).

33. D. Wisser and J. Steffes, Burns. 29(4), 375–380 (2003).

34. G.O. Gey, W.D. Coffman and M.T. Kubicek, Cancer Research 12, 164 (1952).

Mark P. Lewis PhD is director of research operations and senior lecturer at UCL Eastman Dental Institute, UK.

Robert Sexauer is vice president for corporate development at Isolagen Eurpoe Ltd, UK.