Growth Of Human Corneal And Epithelial And Stromal Fibroblast Cells In Serum - Free Media

S.F.Webb
School of Biological Sciences,
University of East Anglia,
Norwich NR4 7TJ
UK

E-mail: s.f.webb@uea.ac.uk

Introduction
Materials
Methods
Results
Summary and conclusions

Introduction

Human Corneal Cell cultures are successfully generated in our laboratories from un-transplantable corneo-scleral discs of all donor ages. Pure epithelial and fibroblast cells are grown routinely in EMEM supplemented with 20% FCS. Although effective for cell growth, the constituents in FCS are neither all identifiable nor quantifiable. As these cells will be used for miniature corneal constructs for growth and toxicity studies, it is preferable to use defined serum-free (SF) media where the constituents can be manipulated.

  THE PURPOSE OF THIS STUDY IS TO FIND SERUM-FREE 
     MEDIA FOR THE ROUTINE GROWTH OF HUMAN CORNEAL CELLS

Materials

Tissues:

Serum-free media tested (Clonetics):

1. KBM Keratinocyte Basal Medium
2. BEBM Bronchial Epithelial Cell Basal Medium
3. MEBM Mammary Epithelial Basal Medium
4. PrEBM Prostate Epithelial Cell Basal Medium
5. REBM Renal Epithelial Cell Basal Medium
6. SABM Small Airway Cell Basal Medium

All these media required several or all of the growth supplements listed below:
Insulin, retinoic acid, hydrocortisone, transferrin, epinephrine, hEGF epidermal growth factor, BPE (bovine pituitary extract) and T3 (tri-iodothyronine).

Methods

A. Observational Studies

1. The epithelial and stromal layers (Fig. 3) were separated by selective sectioning with a novel mini-microtome (Figs.1+2).
2. Each sectioned corneal disc was quartered.
3. The epithelial explants were grown on collagen TC wells.
4. The stromal explants were grown on standard plastic TC wells.
5. Each SF medium was tested at least X10.
6. The control medium was EMEM with 20% FCS.
7. The explants were left undisturbed for 4 days before the first media change.
8. Thereafter the media was changed every 3 days or as growth required till culture confluency.

The criteria of culture success were the speed of culture initiation and the growth to confluency as compared with the control in FCS medium.

Fig. 1		Fig. 2
View of Microtome from the Top		Microtome in cutting Position

Fig. 3

Serial sections from Human Cornea

B. Quantitative Studies

1. Cells were seeded at 3000/200µl of selected medium in each well of a 96-well plate.
2. Each medium was replicated x16 with 4 wells in 4X 96-well plates.
3. Cells were left to grow for 4 days in the incubator with 1 change of medium on day 3.
4. On day 4, cell numbers were determined by the estimation of their total protein content using an absorbance colorimetric method (FRAME Kenacid Blue Protocol 3b)

Results

A) Observational Studies

Epithelial Cultures (Fig 4)

• All the media sustained the initiation of cultures from the explants.
• There was variation in the time taken to establish growth to confluency.
• Cultures in SAGM, PrEGM and REGM took over 3 weeks by which time many cells showed In vitro senescence.
• KGM, BEGM and MEGM achieved confluency within 10-15 days.
• Rank Order of Success in the media: KGM = BEGM -> MEGM -> SAGM = PrEGM -> REGM

Fig. 4

Stromal Fibroblast Cultures

• There was no significant difference in the rate of cell growth amongst the cultures in the different media.
• Initiation was usually 4-7 days after set-up.
• Growth was thereafter rapid and confluency reached within the following 3-4 days.

 Epithelial Cultures (Click for an enlarged image):

2 Day-old culture	10 Day-old culture

Fibroblast Cultures (Click for an enlarged image):

7 Day-old culture	11 Day-old culture

B: Quantitative Studies on P=1 Cells

Fig. 5

Summary and Conclusions

• Corneal stromal fibroblasts thrive in all the serum-free media tested whether as primary or passaged cells.
• 20%FCS containing rich growth promoters in the medium promoted initiation of epithelial cell growth in the explants more rapidly than in 4 of the SF-media.
• However in the P=1 cultures, rather surprisingly, all the serum-free media produced faster growth. Once cell growth is established the need for high concentrations of FCS is usually unnecessary.
• It may actually be detrimental to some cultures as FCS is known to contain also chalones.
• This could be the explanation in this instant. KGM and BEGM are the most effective SF media in supporting corneal epithelial cell growth.

  At present KGM is the medium of choice as BEGM requires
     more supplements as well as at twice the concentration.

Present work:
Human Corneal Constructs
2 methods of assembly are being investigated:

1. Epithelial cells overlay a scaffold of stromal fibroblast/collagen matrix.
2. Multiple sections of stroma produced by the microtome are overlaid with epithelial cells.

Acknowledgements:
Supported by The Humane Research Trust and the RSPCA.
Human corneas supplied by the Bristol Eye Bank.

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