HESC Growth Optimization

One of the challenges we faced during our project was our HESC cells growing very slowly. This made it very hard for us to do experiments, such as transfection, on them. This was a big challenge for us, due to the limited timeframe of the project. When the growth of the HESC cells was first begun, we followed the protocol and growth conditions given by the company we purchased it from (See: “Taking care of HESC cell line” here), but after a little over a month of slow growth, we realized that if we wanted results for the project, we needed to do something about their growth rate.

Observations

A general rule of thumb is that cells are split about every 3 days and that the confluency of the cells needs to be between 70-90% confluency for them to be qualified for a split. Below are the first few entries from our “cell diary” regarding the HESC cells - the notebook was where we noted when the cells were split and how high their confluency was, amongst other things (See the whole notebook here).

NOTE: It is very important to mention that the qualitative data ahead is based on our perception of the cell’s confluency and it should be interpreted with caution. More objective measurement methods could result in a different confluency (%). *Every red line in a graph visualizes when the cells were split

27th of July 2024
Participants: Lina Malou Sörensen (supervisor)
Goal for today: Checking in on the 12Z cells
Cell-line: 12Z
There are more dead cells than usual, presumably due to the late split.
Otherwise, the cells appear healthy, with confluency at 40%.

Cell line: HESC
Protocol: Taking care of 12Z cells
Checking on the newly initiated HESC cell line.
The cells appear alive but are not very confluent, with confluency at 20%.

The note was made on the 27th of July, which was when the first HESC cell line was started, going forward this date will be used as the starting point. Compared to the 12Z cells, the HESC cells had a slow start, additionally after a few days of growth they were maximally 20% confluent.

10th of August 2024
Participants: Susan and Lina Malou Sörensen (supervisor)
Goal for today: Move newly started HESC cells from T25 to a T75.
Make freeze stocks of both cell types
Cell line: HESC (2)
Protocol: Taking care of HESC cells
The cells are moved from a T25 flask to a T75 flask.

Cell line: HESC (1)
Protocol: “Taking care of HESC cells” & “Freezing Eukaryotic Cells”
The cells are split, two freeze stocks are made, and a new flask is started.

Cell line: 12Z
Protocol: “Taking care of 12Z cells” & “Freezing Eukaryotic Cells”
Cells are split, three freeze stocks are made, and a new flask is started.

As noted above we decided it was necessary to start up another flask (referenced to as HESC (2)), as the original flask of cells (referenced to as HESC (1)), was growing slowly. From the starting point, it was not until the 10th of August that they were confluent enough to be split - meaning that it took 14 days of growth and media change, for the cells to be confluent enough to split. In comparison, our original flask of 12Z cells had already been split 4 times in the same time period.

20th of August 2024
Participants: Kathrine & Christina
Goal for today: Determine if cells must be split or require a media change and transfect.
Cell line: HESC (1)
Protocol: “Taking care of HESC cells” & “K2 transfection protocol”
Confluency is at 60% which is too low for splitting. The media is changed.
The flask is combined with HESC (2) to increase growth.
The cells are split in a ratio of 1:10 by accident.
Some are used for K2 transfection with our Control Plasmid.

It took another 10 days for the cells to be split again - and this time they were not even qualified to be split. We decided to combine the two flasks of HESC, to try and increase their growth, as the second flask we started up was growing slow as well. The combined HESC cells were then accidentally split 1:10 instead of 1:3, although this did not seem to have any effect on their growth - which were first qualified for a split in another 10 days on the 1st of September.

(14 days + 10 days + 10 days) 3 splits = 11.3 days

The average time between splits in the original HESC cell line was 11.3 days before we began our growth experiment.

Figure 1: This figure shows a graph containing data from when the HESC cells were first started in the lab. On the x-axis, the time from freshly thawed cells to being confluent enough to split can be seen in the number of days. On the y-axis, the confluency of the cells is shown from 0-80%*.

As can be seen in Figure 1 above, the original HESC cells were growing very slowly under their standard conditions: a T75 flask with DMEM-F12 media with 10% charcoal-stripped FBS, 1% Pen-strep, and 1% L-glutamine. Referring to the previously mentioned rule of thumb, which is that you usually split your cells every 3 days, when they are about 70-90% confluent - we figured that something was off. The original HESC cells had a doubling time of around 8 days, as can be seen from this calculation:

HESC (T₇₅): T_d = 16 days · ln(2) ln(^70%⁄_20%) = 16 days · 0.693 ln(3.5) = 8.87 days

Figure 2: This figure shows a graph containing data from when the HESC cells moved from a T75 flask to a T25 flask, in an attempt to increase the rate of growth. On the x-axis, the time from freshly thawed cells to being confluent enough to split can be seen in the number of days. On the y-axis, the confluency of the cells is shown from 0-100%*.

From Figure 2 it can be seen, that moving the HESC cells from a T75 flask, which has an area of 75 cm2, to a T25 flask, which has an area of 25 cm², had a small effect on the growth rate of the cells. Despite it not looking like a big change, the doubling time of the cells was reduced to 3 days, instead of the original 8, as can be seen in the following calculation.

HESC (T₂₅): T_d = 9 days · ln(2) ln(^90%⁄_20%) = 9 days · 0.693 ln(4.5) = 6.24 days

Based on this, we decided that from that point we were only going to cultivate HESC cells in the T25 flasks, to ensure that they kept up their faster growth rate - but we wanted to see if we could increase it even more.

The Setup

Figure 3: This figure illustrates how the growth optimization experiment was set up. 5 flasks, each containing a different combination of media, were created from the original HESC cells.

Table 1: This table gives an overview of the different media combinations used in the experiment. Each flask was assigned a code (e.g. O2), corresponding to the type of media that it contained.

Type	Conditions
Normal	DMEM-F12 + 10% charcoal-stripped FBS + 1% Pen-Strep + 1% L-glutamine
O1	DMEM-F12 + 10% Human AB Serum + 1% Pen-Strep + 1% L-glutamine
O2	DMEM-F12 + 10% charcoal-stripped FBS + 0.5 % Pen-strep + 1% L-glutamine
O3	DMEM-F12 + 10% FBS + 1% Pen-Strep + 1% L-glutamine
O4	EmbryoMax + 10% charcoal-stripped FBS + 1% Pen-Strep + 1% L-glutamine
O5	EmbryoMax + 10% Human AB Serum + 1% Pen-Strep + 1% L-glutamine

Results

Figure 4: This figure shows a graph containing data for the growth of HESC cells under O1 conditions (see table 1). On the x-axis is the time from right after a split to being confluent enough for another split, which can be seen in the number of days. On the y-axis how confluent the cells are from 0-100%*.

As can be seen in Figure 4, adding 10% Human serum to the media instead of 10% charcoal-stripped FBS had a positive effect on the growth rate of the HESC cells. This could be because Human serum contains several growth factors, such as Platelet-derived growth factor (PDGF), which plays a big role in stem cell regulation (1) and the growth of multipotential stromal cells (2). FBS also contains several growth factors, and even PDGF, but has been known to vary from batch to batch, which could be a possible explanation for the difference in growth rate. Another disadvantage of FBS is the high-dose dependency for in-vitro growth (3). The doubling time was decreased by 6.74 days, when compared to the original HESC cells in a T75 flask, which had a doubling time of 8,87 days.

HESC (O1): T_d = 6 days · ln(2) ln(^70%⁄_10%) = 6 days · 0.693 ln(7) = 2.13 days

In the following days, it was noted that the O1 media looked dirty, and the possibility of contamination of the media was investigated. The test showed no contamination, but the cells looked anomalous, so they were discarded, and as a precaution the media was no longer used.

Figure 5: This figure shows a graph containing data on HESC cells under O2 conditions. On the x-axis, the time from right after a split to being confluent enough for another split can be seen in the number of days. On the y-axis how confluent the cells are from 0-100%*.

As can be seen in Figure 5 the O2 media contained less Pen-strep, with only 0.5% in the media solution instead of the original 1% had a positive effect on the cell growth as well, decreasing the doubling time by 6.32 days instead of the original 8.87 days.

HESC (O2): T_d = 8 days · ln(2) ln(^87.5%⁄_10%) = 8 days· 0.693 ln(8.75) = 2.55 days

Pen-strep is used in cell culturing to prevent contamination with bacteria, but researchers have been starting to second guess the use of it. Pen-strep has been found to harm the cell’s metabolism, especially in immortalized cells. Using antibiotics in cell cultures could also lead to changes in gene expression and regulation (4).

Figure 6: This figure shows a graph containing data on HESC cells growing under O3 conditions. On the x-axis, the time right after a split to being confluent enough for another split can be seen in the number of days. On the y-axis how confluent the cells are from 0-80%*.

As can be seen in Figure 6 the cells growing in the O3 media showed the biggest improvement of all, with a decrease of 6.74 days in their doubling time, compared to the original 8.87 days.

HESC (O3): T_d = 6 days · ln(2) ln(^70%⁄_10%) = 6 days · 0.693 ln(7) = 2.13 days

The only change in the media was that normal FBS was used instead of charcoal-stripped FBS. Normal FBS contains several steroids and growth factors - which have been removed from the charcoal-stripped FBS, by treating it with activated carbon. Charcoal-stripped FBS is usually used to study the effect that steroids have on cell cultures. Since estrogen is a steroid hormone, and the HESC cells originate from the place in the body - the endometrium of the uterus (5) - which is strongly controlled by estrogen, it makes sense that removing such a huge factor from the cell’s media would harm their growth. By “adding it back”, we succeeded in increasing the growth of the HESC cells.

Figure 7: This figure shows a graph containing data on HESC cells growing under O4 conditions. On the x-axis, the time right after a split to being confluent enough for another split can be seen in the number of days. On the y-axis how confluent the cells are from 0-100%*.

By switching the cell culture media from the recommended DMEM-F12 to the media that we used for the 12Z endometrial cells, the EmbryoMax media, a noticeable increase in the HESC cell growth can be seen in Figure 7. DMEM-F12 is a basic medium used for many different mammalian cell types, such as fibroblasts and human endothelial cells. EmbryoMax, on the other hand, is most commonly used for cultivating embryonic stem cells, and since the HESC cells are Human Endometrial Stromal (stem) Cells, this had the potential to serve as a substitute for DMEM-F12. EmbryoMax contains factors such as Krebs-Ringer bicarbonate (KSOM), which helps maintain the pH of the cell culture both in and out of the CO2-incubator. Besides this KSOM also helps stimulate a higher rate of cell division and efficiency. By switching the media to EmbryoMax, we decreased the doubling time of the HESC cells by 7.09 days, as opposed to the 8.87 days for the original HESC cells.

HESC (O4): T_d = 5 days · ln(2) ln(^70%⁄_10%) = 5 days · 0.693 ln(7) = 1.78 days

Figure 8: This figure shows a graph containing data on HESC cells growing under O4 conditions. On the x-axis, the time right after a split to being confluent enough for another split can be seen in the number of days. On the y-axis how confluent the cells are from 0-100%*.

The O5 media contained Human AB Serum, instead of charcoal-stripped FBS. As mentioned earlier, Human AB Serum contains several growth factors, such as Platelet-derived growth factor (PDGF), which plays a big role in stem cell regulation (1). The combination of the EmbryoMax media, specialized for embryonic stem cells, and the growth factors from the Human AB Serum, could be the reason why the use of the O5 media resulted in remarkable growth optimization of the HESC cells as can be seen in Figure 8, decreasing their doubling time by 6.63 days, instead of the original 8. days. In Figure 8 we decided to include the growth of HESC cells in O5 media over 2 rounds of splits, due to the fact that they were growing so quickly.

HESC (O5): T_d = 9 days · ln(2) ln(^90%⁄_14%) = 9 days · 0.693 days ln(1.86) = 2.24 days

Conclusion

In conclusion, the slow growth of HESC cells under standard conditions, such as using DMEM-F12 with charcoal-stripped FBS, presented a significant challenge in the project timeline. However, by optimizing key aspects of the culturing process—such as switching to a smaller T25 flask, replacing charcoal-stripped FBS with regular FBS, and reducing Pen-Strep concentration—we saw considerable improvements in growth rates.

The most dramatic acceleration in growth was achieved by using Embryomax media with 10% charcoal-stripped FBS, which already contains essential growth factors like PDGF. This adjustment resulted in a significant decrease in doubling time, demonstrating that tailoring media more specifically to the needs of stem/stromal cells can greatly enhance growth efficiency, when working with HESC cells.

This information can be valuable for future iGEM teams or researchers working with HESC cells, offering practical strategies for improving cell culture conditions. By leveraging optimized media like EmbryoMax, they can accelerate experimental timelines and improve overall productivity when working with slow-growing cell lines such as HESC. This insight could possibly help streamline research efforts and achieve faster, more reliable results in projects involving these types of cells. If we were to continue with the culturing of HESC cells, outside the frames of this project, we would definitely move forward with using mainly the O4 media, as well as O1 and O3 media, as these all showed the biggest improvement out of all the different media combinations - perhaps one could even further experiment with combining the 3 types of media: EmbryoMax + 10% FBS and/or Human AB Serum + 1% Pen-strep + 1% L-glutamine.