Determination of Superior Culture Conditions for HDF Cells BIOE /28/08

Transcription

1 Determination of Superior Culture Conditions for HDF Cells BIOE /28/08 1

2 Objective Given a range of initial culture factors, determine the most optimal condition for maximum HDF tissue culture viable population Tissue culture factors to be varied: Cell Seeding Concentration Media Concentration of FBS Presence of Attachment Enhancing Treatments 2

3 Experimental Approach Experimental Variable Cell Seeding Concentration Method 1) MTT Viability Test Media Concentration of FBS 2) Cell Proliferation Assay Presence of Attachment Enhancing Treatment 3) Qualitative Fibronectin Attachment Assay 4) Quantitative Cell Attachment Assay 3

4 MTT Viability Test Cells seeded at varying dilutions of ~50,000 cell/ml on 24-well plate 1:1, 1:1.5, 1:2, 1:3, 1:6, 1:12 dilutions in DMEM 10% FBS 1% ab used Control of pure media used Cells incubated for 2h and dyed with MTT Viability assessed by sample absorbance Spectrophotometer measured absorbance at 570 nm MTT absorbs when activated by functional cellular mitochondria Incubation Load into cuvette Trypsin Measure absorbance Figure 1: MTT Viability Test 4

5 Cell Proliferation Assay Cells seeded in 1%, 5%, and 10% FBS DMEM at ~5,000 cell/ml Conditions run in triplicate, and checked after 1, 3, and 7 days Sample in 1% FBS checked at Day 0-4h to establish baseline attachment count Media changed at each sample assessment Proliferation quantified by sample doubling rate Coulter Counter reading of trypsinized cell samples used to determine doubling rate 5

6 Qualitative Fibronectin Attachment Assay Cells seeded onto wells with varying degrees of fibronectin (Fn) saturation Cells added in pure DMEM at ~50,000 cell/ml, run in triplicate Well conditions: A: Control (no Fn): B: Half Fn/Half Control: C: Fn Pattern: D: Fn Saturated: Fn applied by fine paintbrush (B,C) or solution (D) Attachment assessed by observations of morphology, adhesion patterns, and presence of detached cells following 2h incubation 6

7 Quantitative Cell Attachment Assay.1 cm Cell seeded onto untreated, TCtreated, and Fn-coated 24-well polystyrene plates Cells seeded at ~10,000 cell/ml in DMEM (10% FBS, 1% ab) Attachment assessed at 0:30h, 1:15h, 2:30h, and 4:00h All time points run in triplicate Attachment assessed by calculated cell density Cell count performed on 0.01 cm 2 area Density extrapolated assuming representative area Figure 2: Determination of Cell Density 7

8 Statistical Data Analysis Quantitative experiments with multiple data sets were assessed for statistical significance: Experiment Data Sets Sample Size Statistical Test Performed Cell Proliferation Assay 1) 1%, 5%, 10% FBS n = 3 - ANOVA - Tukey s HSD Test Quantitative Cell Attachment Assay 1) Untreated, TCtreated 2) Fn-coated, TCtreated* n = 3 - One-tailed t-test *Data set taken from XXX, and thus assessed independently All tests were passed by the data sets P values are used to quantify significance in results 8

9 Larger Seeding Cell Concentration Produces Larger Viable Cell Culture Population Results of MTT Viability Test: Increased initial HDF cell concentration increases viable culture population linearly without visible saturation effect in measured range Absorbances adjusted to control value = 0 A; R 2 =

10 Increased FBS Concentration in Media Increases Proliferation Rate of Cell Culture Results of Cell Proliferation Assay: FBS conc. 10% Doubling Time 2.9 Days 5% 4.1 Days 1% 6.6 Days Note: Exponential fits used with R 2 > 0.55; Day 7 data excluded due to cell concentration plateau Cell proliferation is more extensive in increased concentrations of FBS The highest tested FBS concentration (10%) had the smallest cell population doubling time Data shown as mean +/- standard deviation; p<

11 Fibronectin Treatment Increases HDF Initial Extent of Attachment Results of the Qualitative Fn Attachment Assay: Condition % Confluence of Attached Cells % Unattached Cells Morphology Observations Untreated (control) <10% 50% Predominantly compact, rounded cells Cell attachment is sparse, present only in small clusters Half Fn/Half Untreated Fn: 20-30% Untreated: <10% Fn: <10% Untreated: >80% Fn: 75% flattened cells, extended pseudopodia Untreated: ~100% compact, rounded cells Clear divide between the two halves in confluence and morphology Fn Design 20-30% (in design regions) 50% Flattened cells with extended pseudopodia found exclusively in patterned regions -Pattern not clearly defined as letters -Regions outside pattern resemble control Fn Saturated 30-40% <10% 90% flattened cells with developed primary pseudopodia Cell size increase of 3-4 fold over control cells Overall, fibronectin proves useful in increasing attached confluence and promoting attached cells to adapt normal morphology Liquid saturation produced better results in general than painted Fn 11

12 TC-treated Plates Promote Increased Cell Attachment Results of Quantitative Cell Attachment Assay: TC-treated plates more than double the extent of cell attachment at all time points Data shown as mean +/- standard deviation; p <

13 Fibronection-coated Plates Promote Further Cell Attachment Results of Quantitative Cell Attachment Assay ** Fibronectin proves to be a statistically significant improvement in promoting attachment as compared to TC-treatment Data shown as mean +/- standard deviation; p < 0.05 **Data taken from XXX 13

14 Fibronectin Positively Impacts Cell Attachment The Qualitative and Quantitative Attachment Assays found that fibronectin increases cell attachment 4h post-seeding in culture Both indicate a roughly two-fold increase in attachment Observation in both cases noted more rapid development of attached morphology Assays had differences in assessing attachment Qualitative: confluence by observation alone Quantitative: cell density by counting Differences in variable measured and qualitative/quantitative method of measurement make direct comparisons between data more difficult 14

15 Conclusion: Modulation of Cell Culture Conditions Increases Success of Culture Within this experiment s range of initial culture conditions, decisions can be made to promote superior extent of attachment, density, and viability of HDF cell culture: Seeding concentration: 50,000 cell/ml Media serum concentration: 10% FBS Well plate treatment: Fibronectin-coated More research necessary to draw conclusion concerning upper limits of optimized conditions Increase FBS and seeding cell concentration further Alternatives exist for increasing cell culture performance 5% FBS, TC-treatment both show significant increases in measured culture variable over controls 15

16 Figure References Figure 1 Well plate: Spectrophotometer: Figure 2 Light microscope: HDF cell culture: 16