[methods in molecular biology] basic cell culture protocols volume 946 || cocultivation of human...

423

Chapter 27

Cocultivation of Human Oral Keratinocytes and Human Osteoblast-Like Cells

Ricarda Glaum and Margit Wiedmann-Al-Ahmad

Abstract

Head and neck reconstruction transplants often require a bony structure but also tissue for the intraoral lining. This is why oral keratinocytes and osteoblast-like cells are essential cell types for combined tissue engineered transplants for defects in the fi eld of craniomaxillofacial surgery. Therefore, we isolated oral keratinocytes and osteoblast-like cells from human tissue samples and cocultivated both cell types on the same carrier. Cell proliferation and morphological analysis showed that the contemporaneous cultivation of human oral keratinocytes and human osteoblast-like cells is possible.

The successful in vitro cocultivation of hard and soft tissue derived cells on the same carrier will be an important advancement for developing hard and soft tissue reconstruction therapies especially in the oral cavity.

Key words: Human oral keratinocytes , Human osteoblast-like cells , Cell cocultivation , Tissue engineering , Cell proliferation , Cell morphology

In head and neck reconstruction, combined hard and soft tis-sue reconstructive therapy is often necessary. Besides the bony reconstruction which is addressed by different approaches, in many cases an intraoral lining by oral mucosa is required ( 1– 5 ) . Therefore, ideal transplants for combined hard and soft tissue reconstruction by the means of tissue engineering approaches require the combination of different cell types. We investigated the cocultivation of human oral keratinocytes and human osteoblast-like cells as common cell types present in head and neck region. The cells were isolated from bone and oral mucosa samples, obtained during oral surgical procedures, by direct explant technique. In contrast to the commonly used technique of culturing single cell suspensions, the tissue

1. Introduction

Cheryl D. Helgason and Cindy L. Miller (eds.), Basic Cell Culture Protocols, Methods in Molecular Biology, vol. 946, DOI 10.1007/978-1-62703-128-8_27, © Springer Science+Business Media, LLC 2013

424 R. Glaum and M. Wiedmann-Al-Ahmad

explant technique has been shown to be more effective for oral keratinocytes as well as for osteoblast-like cells ( 6– 9 ) .

We have shown that the simultaneous cultivation of these two cell types is possible. Cells can be cultivated in culture dishes as well as on different membranes. There are many factors that in fl uence the result of the experiments including contamination of the patient’s samples with oral micro fl ora, contamination of the keratinocytes with fi broblasts and as well as different cell cycles times of the kera-tinocytes and osteoblasts. Furthermore, the proliferation analysis of cocultivated cells within the same culture shows lower levels of via-bility than one would expect compared to the separate cultivation of each cell type ( 10 ) . It is possible that the different cell types hinder the proliferation of each other. A barrier between the cells has been shown to enhance their viability ( 11 ) . Different carriers vary in their ability to support cocultivation, and changing culture conditions (for example by using perfusion culture) does not necessarily result in the same advantages for cocultivation when compared to cultiva-tion of only one cell type ( 12, 13 ) . In this chapter, we describe meth-ods for the isolation and culture of oral keratinocytes and osteoblast-like cells isolated from human tissue samples.

1. Dulbecco’s Phosphate Buffered Saline (PBS, PAA Laboratories, Linz, Austria), store at room temperature (RT).

1. Opti-MEM1 with Glutamax (Opti Minimal Essential Medium, Gibco Invitrogen Life Technologies, Paisley, UK). Store at 4–8°C.

2. Fetal calf serum (FCS, PAA Laboratories, Linz, Austria). Inactive FCS at 56°C for 30 min and store in aliquots at −20°C.

3. 1 M N-2-hydroxyethylpiperazine-N ¢ -2-ethanesulfonic acid (HEPES, Roth, Karlsruhe, Germany), store at room temperature.

4. CaCl 2 (Roth, Karlsruhe, Germany), mix the powder with high grade water, and sterilize by autoclaving. Store at room tem-perature for up to 1 year.

5. Penicillin/streptomycin solution (PAA Laboratories, Linz, Austria). Store aliquots at −20°C.

6. Osteoblast-like cell medium: To prepare medium combine components to fi nal concentrations of:

Opti-MEM1 with Glutamax, 10% FCS, 2% HEPES (0.02 M HEPES), 1,000 mg/L CaCl 2 , and 1% penicillin/streptomycin. Store at 4–8°C for up to 3 weeks.

2. Materials

2.1. Medium for Cultivation of Osteoblast-Like Cells

42527 Cocultivation of Human Oral Keratinocytes and Human Osteoblast-Like Cells

1. Dulbecco’s Modi fi ed Eagle Medium (DMEM) with 1 g L-Glucose (Bio Whittaker, Cambrex Bio Science Verviers, Verviers, Belgium), store at 4–8°C for up to 3 weeks.

2. Nutrient Mixture Ham’s F-12 with Glutamine (Gibco Invitrogen Life Technologies, Paisley, UK), storage at 4–8°C for 3 weeks.

3. L-glutamine (PAA Laboratories, Linz, Austria), thaw and store in aliquots at −20°C.

4. The following reagents are dissolved in DMEM:F12 medium and fi lter-sterilized using a 0.2 micron low protein binding fi lter. Prepare aliquots suf fi cient for 1 L of medium and store at −20°C. (a) Epidermal growth factor (Sigma, Roedermark, Germany

(10 m g/L)). (b) Insulin (from bovine pancreas, Sigma, Roedermark,

Germany) (5 mg/L). (c) Human transferrin (apo-transferrin, Sigma, Roedermark,

Germany) (5 mg/L). (d) Triiodotyronine (Sigma, Roedermark, Germany) (1.36 m g/L). (e) Hydrocortisone (Sigma, Roedermark, Germany)

(0.2 mg/L). (f) Cholera toxin (Sigma, Roedermark, Germany)

(0.0085 mg/L). (g) Adenine (Sigma, Roedermark, Germany) (0.024 g/L). (h) Spermine (Sigma, Roedermark, Germany) (10.5 mg/L).

5. To prepare oral keratinocyte medium combine: 750 ml DMEM and 250 ml nutrient mixture HAM’s F12, and

aseptically add 10% FCS, 2% HEPES (0.02 M HEPES), 1% penicillin/streptomycin, 10 micrograms/L EGF, 5 mg/L insulin, 5 mg/L transferrin, 0.0085 mg/L cholera toxin, 0.024 g/L adenosine,10.5 mg/L spermine

1. 0.5% trypsin in PBS (PAA Laboratories, Linz, Austria), Thaw overnight in refrigerator, aliquot, and store at −20°C. Dilute in PBS as needed for different cell types.

1. RPMI 1640 (Gibco Laboratories Life Technologies, NY, USA), supplemented with10% FCS, 1% penicillin/streptomy-cin solution, 5% HEPES ( fi nal concentration). Store at 4–8°C for up to 3 weeks.

1. 100 m m cell strainer, 50 ml tubes (Falcon, Heidelberg, Germany). 2. Culture fl asks; 25 cm 3 and 75 cm 3 , (Greiner, Frickenhausen,

Germany).

2.2. Medium for Cultivation of Oral Keratinocytes

2.3. Cell Trypsinization

2.4. Medium for Cell Cocultivation

2.5. Cell Culture Supplies


3. Carriers for cocultivation. Polycarbonate membranes (Millipore, Minucells and Minutissue, Bad Abbach, Germany, pore size 0.4 m m, diameter 13 mm) as well as equine collagen membranes (Tissue Foil E ® , Baxter, Resorba, Nürnberg, Germany) were used.

Samples of bone and oral mucosa are harvested from patients (for example during oral and maxillofacial interventions) under sterile conditions and placed into sterile 0.9% NaCl solution ( see Note 1 ). The different samples are processed separately. For isolating the cells out of the samples, we use the direct explant technique ( 6– 9 ) .

1. For the cultivation of osteoblast-like cells samples of bone are rinsed with ethanol (70%) for 10–20 s, then rinsed three to four times with PBS and minced into pieces of 1–2 mm diameter.

2. Culture fl asks (25 cm 3 ) are fi lled with 1.5 ml osteoblast-like cell medium ( see Subheading 2.1 ). Place three to fi ve pieces of sample into each culture fl ask.

3. Incubate explant cultures at 37°C in humidi fi ed atmosphere with 5% CO 2

4. After 1 week add 1 ml medium, another 1 ml 3–4 days later and 3–4 days later 1.5 ml medium is added (~5 ml total volume per fl ask).

5. The osteoblast-like cell medium is changed every 3–4 days until the adherent cells cover the bottom of the culture fl ask.

6. For the fi rst passage the con fl uent cell culture (primary cul-ture) are detached from the culture fl ask by incubation with 0.5% trypsin in phosphate buffered saline for 8 min at 37°C.

7. The cell solution is fi ltered through a 100 m m cell strainer in 50 ml tube, centrifuged at 1120 × g , 12 min at 30°C and resus-pended in 1 ml culture medium.

8. Cells are placed in a 75 cm 3 culture fl ask with cocultivation medium ( see Subheading 2.3 ) and incubated.

9. After reaching con fl uence again the cells are detached from the culture fl ask by incubation with 0.5% trypsin in phosphate buffered saline for 8 min at 37°C, centrifuged, resuspended in 1 ml medium and cultivated (second passage).

10. Cells are used for experiments after second passage ( see Note 4 ). To verify the cell type, a portion of the cells should be cultivated, e.g., for 1 week separately and used for cell characterization ( see Note 5 ).

3. Methods

3.1. Isolation and Cultivation of Cells

3.2. Isolation and Cultivation of Osteoblast-Like Cells


1. For the cultivation of oral keratinocytes samples of mucosa are rinsed with ethanol (70%) for 10–20 s and afterwards three to four times with PBS, minced into pieces of 1–2 mm diameter. For pure cultures of keratinocytes it is crucial to separate the epithelial layer of the sample from the underlying connective tissue using a sharp blade ( see Note 3 ). Otherwise, there is a high risk of contamination with fi broblasts.

2. Place three to fi ve pieces of the sample into culture fl asks (75 cm 3 ) fi lled with 4 ml of medium for cultivation of oral keratinocytes ( see Subheading 2.2 ; Note 2 ).

3. The explants are incubated at 37°C in humidi fi ed atmosphere with 5% CO 2 .

4. The amount of medium is increased step by step to a total vol-ume of 10 ml. After outgrowth of the cells, 30 ml of medium is used for medium change.

5. Cells are used for experiments after the fi rst passage ( see Note 4 ). To verify the cell type a part of the cells should be cultivated (e.g., for 1 week) separately for cell characterization ( see Note 5 ).

1. Separately cultivated cells are trypsinized and seeded into cul-ture dishes or on different carriers. Another medium is used for cocultivation to ful fi ll the demands of both cell types ( see Subheading 2.3 ). It contains a higher amount of HEPES because many experiments are carried out outside the incubator.

2. For experiments using membranes as carriers the cells are placed on the opposite sides of the membranes (diameter of the membranes: 13 mm). Therefore, one cell type is placed onto the carrier on 1 day ( 12 ) . After adherence of the cells the other cell type is placed 1 day later onto the other side of the carrier. We use 1 × 10 5 cells diluted in 50 m l medium of each cell type ( see Note 4 ). Polycarbonate membranes as well as equine collagen membranes were used.

In scanning electron microscopy as well as by light microscopy the cell types can be differentiated by their typical morphology and growth-pattern: Osteoblast-like cells are longish cells arranged like a draught of fi shes in con fl uent layers. Oral keratinocytes are compact, polygo-nal, fl at and show a cobblestone pattern when con fl uent (Fig. 1 ).

1. For all cell culture experiments it is essential to work under sterile conditions (e.g., using sterile media and solutions, work-ing under laminar fl ow).

3.3. Isolation and Cultivation of Oral Keratinocytes

3.4. Cocultivation

3.5. Cell Morphology

4. Notes


2. Especially the samples for oral keratinocytes should be taken under sterile conditions, e.g., intraoral disinfection with betadine/iodine before taking the biopsy.

3. For the isolation of keratinocytes from mucosa it is essential to separate completely the epithelial layer of the tissue from the underlying connective tissue to avoid contaminations with fi broblasts.

4. For cocultivation of both cell types, it is essential to use the second passage of human osteoblast-like cells and the fi rst pas-sage of keratinocytes because of differences in the growth behavior.

5. Before using the cells for experiments the cell type should be veri fi ed by cell characterization like testing for example osteo-calcin, alkaline phosphatase, collagen type 1 for osteoblast-like cells and cytokeratins type 13 and 19 for oral keratinocytes.

Fig. 1. The typical morphology and growth pattern of the different cell types is visible in scanning electron micrographs of osteoblast-like cells ( a , b ) and oral keratinocytes ( c , d ) on the opposite sides of a collagen membrane (Tissue Foil E ® , Baxter, Resorba, Nuremberg, Germany) after two weeks of cultivation ( 13 ) . Magni fi cation ×200 ( a and c ) and ×1,000 ( b and d ).


References

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5. Terheyden H, Warnke P, Dunsche A et al (2001) Mandibular reconstruction with pre-fabricated vascularized bone grafts using rec-combinant human osteogenic protein-1: an experimental study in miniature pigs. Part II: transplantation. Int J Oral Maxillofac Surg 30:469–478

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8. Jonsson KB, Frost A, Nilsson O et al (1999) Three isolation techniques for primary culture of human osteoblast-like cells: a comparison. Acta Orthop Scand 70:365–373

9. Voegele TJ, Voegele-Kadletz M, Esposito V et al (2000) The effect of different isolation techniques on human osteoblast-like cell growth. Anticancer Res 20:3575–3581

10. Glaum R (2008) Tissue Engineering von Composite Grafts: Cokultivierung von Gingivakeratinozyten und Osteoblasten auf Polycarbonat- und Kollagenmembranen. VVB Laufersweiler Verlag, Giessen

11. Sendic M (2005) Wachstumsverhalten und gegenseitige Beein fl ussung von Gingivakeratino-zyten und Osteoblasten auf Kollagen. Inaugural-Dissertation zur Erlangung des Zahnmedizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i Br

12. Glaum R, Wiedmann-Al-Ahmad M, Huebner U et al (2010) Tissue engineering of composite grafts: cocultivation of human oral keratinocytes and human osteoblast-like cells on laminin-coated polycarbonate membranes and equine collagen membranes under different culture conditions. J Biomed Mater Res A 93:704–715

13. Otto F (2004) Vergleichende Untersuchungen des Wachstums humaner Osteoblasten nach Kultivierung im Brutschrank und in der Perfusionskammer. Inaugural-Dissertation zur Erlangung des Medizinischen Doktorgrades der Medizinischen Fakultät der Albert-Ludwigs-Universität Freiburg i Br, Freiburg

[methods in molecular biology] basic cell culture protocols volume 946 || cocultivation of human...

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