University of New Haven: BIOM 6671 Tissue Engineering Lab Report

This lab report details an experiment on human mesenchymal stem cells (hMSCs) in a tissue engineering context. The aim was to suspend and seed hMSCs at a specific concentration. The methodology involved culturing hMSCs in a basal medium, changing the medium every two days, and incubating the cells at 37°C with 5% CO2. Upon reaching 100% confluency, the cells were trypsinized, centrifuged, and resuspended. ImageJ software was used to analyze cell counts from multiple fields of view, providing data on cell area, mean, minimum, maximum, and coordinate locations. The results section presents the image analysis data. The report includes an introduction to hMSCs, materials and methods, results, discussion, and conclusion, providing a comprehensive overview of the cell culture process and analysis.

Running head: TISSUE ENGINEERING
LAB REPORT
Name of the Student
Name of the University
Author Note

1TISSUE ENGINEERING
Table of Contents
Introduction................................................................................................................................2
Aim and objectives.....................................................................................................................2
Aim.........................................................................................................................................2
Objectives...............................................................................................................................3
Materials and method.................................................................................................................3
Materials required..................................................................................................................3
Method used to perform the experiment................................................................................3
Results........................................................................................................................................4
Calculations..............................................................................................................................12
Discussion................................................................................................................................14
Conclusion................................................................................................................................14
Bibliography.............................................................................................................................15

2TISSUE ENGINEERING
Introduction
Human mesenchymal stem cells or hMSC are a particular type of cells which are
characterized by both the self-renewal and the property of pleuripotency. This property has
been found to allow the cells to differentiate into various significant cell types. These cellular
types includes myocytes of muscle cells, osteoblasts or bone cells, adipocytes of fat cells and
chondrocytes of cartilage cells.
The oldest research studies based on stem cells include the findings for the precursor
of mesenchyme cells which is responsible for the development of various types of blood
cells. This discovery was done by a Russian born morphologist Alexander A. Maximov who
used extensive histological findings to give identify the singular type precursor cells as stated
before. The clonal nature of narrow cells were first revealed by two scientists named as
Ernest A. McCulloch and James E. They revealed the clonal nature of stem cells in the year
1960 just after the first discovery was done in 1924. After the previous discovery, an ex vivo
assay for the examination of clonogenic potential of multipotent marrow cells was
significantly reported in 1970. This discovery was again done by a group of scientists named
as Friedenstein and colleagues. CFU-f or the colony forming fibroblasts have been referred to
as stromal cells inside this assay system.
This report will first discuss the aim and objectives of the practical work followed by
stating its methodology to give rise to the results which can be used to calculated the number
of cells per field that will be discussed and concluded based on the findings.

3TISSUE ENGINEERING
Aim and objectives
Aim
The main aim of this practical is to suspend the hMSC’s and then seed them to a
specific concentration.
Objectives
To undergo successful cell seeding using the laboratory protocol stated after this
section.
1. To estimate the field of view from the cell growth images.
2. To estimate the total number of cells present per field of view.
3. To estimate the number of cells per well.
Materials and method
Materials required
Test cells- Human Mesenchymal Stem Cells
Physical equipments- MSCGM Singlequots supplement kit, incubator, centrifuge,
hemocytometer, 1 mL, 5 mL, 200uL, 1000uL pipette tips.
Media and chemicals- MSCBM Basal Media, Trypan blue, 0.25% EDTA trypsin.
Glassware- 12 well sterile plates
Software- ImageJ
Method used to perform the experiment
Cell culture process

4TISSUE ENGINEERING
HMSCs were cultured inside the hMSC5% basal medium after the preparation of
basal medium by medium kit was completed. This culture medium needs to be changed after
every two days. This culture medium along with the cells were incubated inside a 37 degree
centigrade fixed incubator with the administration of 5% carbon di oxide. After the cells each
a level of 100% confluency, the hMSC were then trypozonied by the use of 0.25% EDTA
trypsin. This process was performed for 5 minutes before detaching the cells from the
substrate. Then the cells were detached from the substrate and they were centrifuged at 200G
for 5 minutes before finally aspirating the medium. Then 3ml of mesenchymal stem cell
medium was pipetted up and down thoroughly for ten times. Finally, it can be stated that the
cells were ready to be counted after the addition of 50 microliter of basal medium and 50
microliter Trypan Blue and 15 microliter of hMSC after counting the cells on the four corners
of the hemocytometer and the cellular concentration.
Results
The following figures show an estimating number of the hMSC after culturing for 7 days of
incubation.

5TISSUE ENGINEERING
Area Mean Min Max X Y
1 0 74 74 74 164 60
2 0 74 74 74 164 60
3 0 107 107 107 744 108
4 0 140 140 140 388 108
5 0 140 140 140 388 108
6 0 90 90 90 732 684
7 0 115 115 115 992 244
8 0 115 115 115 992 244
9 0 82 82 82 1544 472
10 0 198 198 198 1728 344
11 0 198 198 198 1728 344
12 0 90 90 90 2152 472
13 0 115 115 115 2172 684
14 0 115 115 115 2172 684
15 0 156 156 156 144 636
16 0 115 115 115 1044 1192
17 0 115 115 115 1044 1192
18 0 140 140 140 1272 308
19 0 99 99 99 1400 124
20 0 99 99 99 1400 124
21 0 115 115 115 1660 552
22 0 107 107 107 2184 1020
23 0 107 107 107 2184 1020
24 0 90 90 90 2104 1160
25 0 90 90 90 2492 424

6TISSUE ENGINEERING
26 0 90 90 90 2492 424
27 0 66 66 66 1756 924
28 0 140 140 140 2068 1816
29 0 140 140 140 2068 1816
30 0 132 132 132 1660 1248
31 0 132 132 132 716 1596
32 0 132 132 132 716 1596
33 0 148 148 148 344 1492
34 0 173 173 173 344 1004
35 0 173 173 173 344 1004
36 0 82 82 82 1624 44
37 0 115 115 115 828 972
Figure 1: Image analysis of hMSC field 1
Area Mean Min Max X Y
1 0 91 91 91 104 72
2 0 91 91 91 104 72
3 0 96 96 96 372 232
4 0 105 105 105 1264 192
5 0 105 105 105 1264 192
6 0 96 96 96 1740 164
7 0 103 103 103 1996 396
8 0 103 103 103 1996 396
9 0 95 95 95 1712 536
10 0 93 93 93 2536 376

7TISSUE ENGINEERING
11 0 93 93 93 2536 376
12 0 93 93 93 2516 548
13 0 95 95 95 2540 784
14 0 95 95 95 2540 784
15 0 95 95 95 1272 668
16 0 88 88 88 640 628
17 0 88 88 88 640 628
18 0 102 102 102 768 1100
19 0 99 99 99 312 1228
20 0 99 99 99 312 1228
21 0 95 95 95 1436 1328
22 0 92 92 92 2036 1360
23 0 92 92 92 2036 1360
24 0 99 99 99 2116 1176
25 0 95 95 95 2436 1576
26 0 95 95 95 2436 1576
27 0 89 89 89 2052 1876
28 0 93 93 93 1096 1184
29 0 93 93 93 1096 1184
30 0 87 87 87 2496 36
31 0 98 98 98 260 1500
32 0 98 98 98 260 1500
33 0 97 97 97 1704 1584
34 0 98 98 98 600 300
35 0 98 98 98 600 300
36 0 94 94 94 460 460
37 0 93 93 93 312 768
38 0 93 93 93 312 768
39 0 93 93 93 1636 780
40 0 68 68 68 1420 1524
Figure 2: Image analysis of hMSC field 2

8TISSUE ENGINEERING

9TISSUE ENGINEERING
Area Mean Min Max X Y
1 0 95 95 95 36 76
2 0 95 95 95 36 76
3 0 92 92 92 344 100
4 0 100 100 100 704 152
5 0 100 100 100 704 152
6 0 96 96 96 1252 492
7 0 91 91 91 1724 200
8 0 91 91 91 1724 200
9 0 90 90 90 1904 68
10 0 92 92 92 2432 548
11 0 92 92 92 2432 548
12 0 87 87 87 2540 496
13 0 88 88 88 2572 56
14 0 88 88 88 2572 56
15 0 94 94 94 2564 808
16 0 95 95 95 2344 728
17 0 95 95 95 2344 728
18 0 101 101 101 1432 1120
19 0 95 95 95 1468 744
20 0 95 95 95 1468 744
21 0 95 95 95 880 908
22 0 81 81 81 460 608
23 0 81 81 81 460 608
24 0 95 95 95 640 308
25 0 96 96 96 948 220
26 0 96 96 96 948 220
27 0 99 99 99 992 504
28 0 98 98 98 1212 400
29 0 98 98 98 1212 400
30 0 101 101 101 1336 224
31 0 96 96 96 1648 468
32 0 96 96 96 1648 468
33 0 97 97 97 1340 1212
34 0 92 92 92 1220 1164
35 0 92 92 92 1220 1164
36 0 97 97 97 1908 1680
37 0 92 92 92 2152 1560
38 0 92 92 92 2152 1560
39 0 101 101 101 2240 1400
40 0 93 93 93 1320 1648
41 0 93 93 93 1320 1648
42 0 100 100 100 1388 1768
43 0 93 93 93 652 1632
44 0 93 93 93 652 1632
45 0 94 94 94 284 1168

10TISSUE ENGINEERING
46 0 98 98 98 140 820
47 0 98 98 98 140 820
48 0 98 98 98 484 1028
49 0 96 96 96 768 1052
50 0 96 96 96 768 1052
51 0 98 98 98 244 268
Figure 3: Image analysis of hMSC field 3
Area Mean Min Max X Y
1 0 96 96 96 60 64
2 0 96 96 96 60 64
3 0 97 97 97 116 240
4 0 98 98 98 212 532
5 0 98 98 98 212 532
6 0 93 93 93 292 872
7 0 101 101 101 312 1040
8 0 101 101 101 312 1040
9 0 101 101 101 328 1336
10 0 94 94 94 692 940
11 0 94 94 94 692 940
12 0 99 99 99 744 888
13 0 97 97 97 536 1032
14 0 97 97 97 536 1032
15 0 102 102 102 500 736

11TISSUE ENGINEERING
16 0 93 93 93 1248 588
17 0 93 93 93 1248 588
18 0 97 97 97 1160 764
19 0 98 98 98 1640 476
20 0 98 98 98 1640 476
21 0 99 99 99 1796 364
22 0 100 100 100 1432 36
23 0 100 100 100 1432 36
24 0 97 97 97 1980 360
25 0 95 95 95 2248 396
26 0 95 95 95 2248 396
27 0 104 104 104 2240 516
28 0 96 96 96 2400 700
29 0 96 96 96 2400 700
30 0 96 96 96 1444 860
31 0 101 101 101 1548 564
32 0 101 101 101 1548 564
33 0 96 96 96 1044 1240
34 0 97 97 97 1316 1256
35 0 97 97 97 1316 1256
36 0 96 96 96 1812 988
37 0 100 100 100 2144 1276
38 0 100 100 100 2144 1276
39 0 106 106 106 2384 1408
40 0 101 101 101 1964 1500
41 0 101 101 101 1964 1500
42 0 102 102 102 476 1740
43 0 94 94 94 340 1904
44 0 94 94 94 340 1904
45 0 99 99 99 172 1832
46 0 95 95 95 2108 92
47 0 95 95 95 2108 92
48 0 96 96 96 2332 44
49 0 96 96 96 568 24
50 0 96 96 96 568 24
51 0 98 98 98 904 396
Figure 4: Image analysis of hMSC field 4