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The original extended story accompaniment for Acquisitions II - Product
SDF – March 6th, 2011.
It has taken a while to get things up and running here in lab group seventeen. Numerous setbacks and shifting of personnel during the holiday season provides no lack of frustration. However, I am rather proud of the results we have achieved in these scant six months. Our collaboration with Dr. Hayes has been fruitful, but quite demanding – although given the number of significant figures on his cheques, his impatience is understandable.
We started out back in late August, trying to apply a wet-growth technique following a rather promising procedure1 I had seen applied back in a conference from ’08. It involved using very high concentrations of cells (relative to the amounts we normally employed) to accelerate the formation of tissues. A slew of authors claimed that jacking up the cell count of the stuff you were starting with would net faster tissue formation. Diagram and diagram pointing towards heightened number of cells raising the expression of CAM’s (cell-adhesion molecules). The theory seemed sound enough; the more sticky cells you had the better they stuck together – but it was fairly suspicious that no one was posting hard number. Which CAMs were being activated? By how much? And most importantly why?
I should have trusted my gut there. The squirming, writhing snake of doubt coiling in my insides as I tried something I’d not attempted before. Would have saved me two and half months of glaring down the eyepiece of a microscope, that’s for sure. Every well or petri gummed up within days of plating. You would see the most beautiful islands of cells form; little raft-like colonies serenely collecting in the growth media. Then it all went sour. The rafts wouldn’t connect on the surface. I wanted an archipelago, but all I got were cancerous little atolls. The keratinocytes would form hideous spheres that choked the cells in the center – starved them of the nutrients on the outside. The resulting dead zones would poison everything outward until you had a thin rim of struggling cells, glued to the corpses of their parent and sister generations. It was frustrating – we switched out growth media. We changed cell lines. Didn’t matter – stick too many cells into a plate at once and you were going to get an epidermal traffic jam.
November brought Hayes’ annoyance clip-clopping around the lab. Finding out what doesn’t work is well and fine for academia but the Hindu horse was not after papers. We had several lengthy discussions before I finally decided to change methodology. To appease his interests I switched to using equine cell lines and a growth medium that was more solid-phase2,3. The method was the one I had originally planned to use but due to the amount of time and effort needed had decided against it. But with Hayes breathing down my neck and three months already exhausted, a few weeks on a more reliable method seemed like a better trade off.
I spent the bulk of late November all the way the brink of January perfecting the method. Over the Christmas holiday I stumbled across an exciting publication4 that greatly accelerated our work as well. I ended up shifting away from wet-methods and commandeered our old crystallography preparation glove box to exploit the sterile environment. Ueda’s method called for repeated spraying of pre-cultured cells onto burn victims our skin – however their process was slow and required months of rehabilitation. Why settle for squirts and dabs, hoping things would take root? Why strap your patients down and tell them not to itch their growing skin when you could present them with their new flesh all at once and skip the acclimation? They were trying to play the odds. They knew half the time it would fail and went with a less work intensive method. More chances to succeed would lessen the brunt of the many failures. They strove for statistical significance. But that’s the mistake with thinking academically. You think 7/10 is a good chance of success – it’s only three duds! – but when you’re dealing with rare materials and peoples lives – that’s three chances for things to go wrong. That three chances you’re going to waste your time and attention. I intended to take chance out of the equation.
Solid phase has the advantage that you can do things in rapid sequence and “high” yield. It makes complex chemistry trivial. It lets you rival enzymes in speed and specificity. It’s a multibillion dollar industry that has made it possible to call someone up and order proteins and custom DNA sequences. It also only works on a select number of substrates and can be finicky at that. If the building blocks are available then people will gladly hop right on that for you. If they aren’t.. well then be prepared to start tooling with a system that took the greater part of the four decades to master. You want a methyl group here? Add an extra zero to pricetag. Thankfully, that wasn’t the case in this situation. All they had to do was stick a couple of peptides off the surface off polyacrylate. That was doable enough – you just dipped your desired surface in this solution for an hour, rinsed and repeated as needed. It left us with a microlayer of peptide sticking up off the surface. A painted sheath of molecular Velcro™ that we could stick whatever we wanted onto.
Our initial tests proceeded marvelously. We covered a polyacrylate plate in CAM analogs so the initial seeding of equine keratinocytes would have someplace to stick. No need to worry about rafts or spheres in solution – they’d form a nice even mat where we wanted. After that we just just applied daily mistings of daughter cells to keep thing moving along. Epidermal cells are easy to work with once you get them going; they don’t require a vascular network to suckle nutrients from. No need for complicated artificial blood vessels. Mix your favorite nutrient broth into the daily spritz and they grow happily. It was kind of like gardening really. A nice, thick mat of the pinkest, healthiest flesh you could imagine, sprawling out, eager for more surface space. The procedure and solution prep was simple enough I could even teach it to the other lab staff. By mid-February we were averaging a quarter of a foot of viable, transplantable skin a week. I was due time, and I decided to send Hayes an update.
He ended up coming in earlier today and I could not have been more excited to show off our progress. We had already switched over to a morphic cell line – taken from a patient in a burn unit here in Colorado. She was a wolf of some uncertain descent – although you wouldn’t know it looking at her. Her right hand, arm, shoulder and face had been severely injured from a grease fire. An industrial sized kitchen deep-fryer had caught aflame and her efforts to put it out with an extinguisher had instead splattered sizzling oil all over the place. Namely her. The file photo was grizzly – a wrist and lower arm devoid of fur and bundled tight with oozing gauze. A muzzle deformed by heat, snout sanded down to the bone by a rough licking tongue of heat. One eye was effectively glued shut by her presently fused eyelids. The damage draped down her neck and neck like a sickening shawl of melted flesh. The burn unit had had to perform an emergency skin graft to repair her throat after it became infected and ended up not using all of the flesh they borrowed from her right thigh. Hayes had convinced them to donate it to our project.
The look of revulsion that crumbled his face and bent the tilaka on his brow spoke volumes regarding the success of our work. It is the look of surprise. The mask you wear when gazing into something unexpected. I have seen that look before. I have worn it before. It is what we scientists adorn ourselves when confronted with an unanticipated discovery. A strange crystal in the bottom of our beakers. A curious color change in solution. A napkin sized hunk of skin dangling off a mannequin. Hayes arms folded as he gawked at the glistening, naked flesh I had put on display for him – as if to put a barrier between himself and the product.
“What is this?” he had managed after a moment, to which I was oh so happy to reply,
“It’s her.”
There was an awkward span of disquiet as Hayes struggled to digest the information. To intake the majesty of what our project had accomplished. I offered further elucidation,
“As you can see, Dr. Hayes, we have been able to successfully grow a tissue culture from our candid collaborator. We’ve transferred cultured cells into solution and have been spray coating them onto a surface so that – “
“Well – yes I can see that,” he rudely interrupted, “But, what is it.. mounted on?”
“Oh! Do you like the mannequin? We needed a solid support for the growing tissue and I decided that, rather than use something mundane that a more dynamic surface would suit. As you can see –”
I paused to wriggle my arms into the black neoprene arm ports of the glovebox. The upset in homeostasis caused a hiss and sputter of escaping pressure to billow from a side port. The whoosing wail compelled an air compressor beneath to whirl into motion. The thu-thu-thunking of the piston forced me to raise my voice over the mechanical symphony as I grabbed the sides of the mannequins head and pivoted it to more directly face us.
“ – the keratinocytes take very readily to the surface! Through careful application, we can grow the developing tissue into any structure we want! We transplanted some of the facial tissue and are trying to grow fingers –”
– I motioned to the hand fragment sporting what amounted to a fingerless glove of wolf-skin.
“I recalled from the patient’s photos, she had experienced damage around the eyes? So what I have done here is drawn some lines. We’re going to inject ceramide1 – and that’ll make the cells here engage in apoptosis,” I picked up a pipette to demonstrate, circling around the black-marker spots a few times for emphasis. “..Bam! Instant eye holes.”
Hayes was silent for a while. I considered repeating myself in case he had not heard me over the pump but his look of disgust had not softened. His gaze stretched across the face of the mannequin – before twisting away, as though he could not bear to meet its eyeless stare. Instead, he scrutinized the hand whose frozen grasp awaited the touch of developing skin. I tried to read his expression, but looking over my shoulder, elbows deep in arm ports was neither comfortable nor useful. I could see something whirling in the gears behind those bent mawari ears. He scanned along the fleshless fingers of the hand, musing on it before looking at the pipets and then back up to me.
“How fast is it growing? Do you think you could have an arm’s worth by April?”
“That might be pushing it, but yeah I think so. Do you want me to send you progress shots?”
“No thank you Dr. Fluttertail – I’ll send you measurements for the patient. I’ll meet with you then,
I frowned a bit, watching him leave in a hurry. I rather expected him to be more… excited about the advances we’d made. The specificity of his request was promising, but his tone was off-putting. Wasn’t this exactly what he wanted? He knew how much work had gone into this project. It was hard to imagine something as visually distasteful as skin growing on mannequin could turn the stomach of a morph whose own daughter had half her flesh sizzled off. Perhaps it was a cultural thing – but even so, one really oughtn’t look a gift horse in the mouth.
1] Deshpande M. “Three-dimensional organization of dermal fibroblasts by macromass culture.” Biotechnology and Applied Biochemistry 2008 49(1) 65-72
2] Lima-Neto JF,; Fernandes CB,;, Alvarenga MA,; Golim MA,; Landim-Alvarenga FC. “Viability and cell cycle analysis of equine fibroblasts cultured in vitro.” Cell and Tissue Banking, 2010, 26(5), 365-368
3] Chen C,; Li GF,; Liu W,; Yang NZ,; Wang B,; Zhang C,; Wang ZJ.; “Construction of Tissue-Engineered skin by mix-seeding” Chinese Journal of Plastic Surgery 2010, 26(5), 365-368
4] Ueda M, “Sprayed Cultured mucosal epithelial cell for deep dermal burns,” Journal of Craniofacial Surgery 2010, 21(6), 1792-1832
5] Tarek A.T,; Thomas D.M,; Lina M.O; “A house divided: ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death,” Biochimica et Biophysica Acta 2006 12, 2027-2036
SDF – March 6th, 2011.
It has taken a while to get things up and running here in lab group seventeen. Numerous setbacks and shifting of personnel during the holiday season provides no lack of frustration. However, I am rather proud of the results we have achieved in these scant six months. Our collaboration with Dr. Hayes has been fruitful, but quite demanding – although given the number of significant figures on his cheques, his impatience is understandable.
We started out back in late August, trying to apply a wet-growth technique following a rather promising procedure1 I had seen applied back in a conference from ’08. It involved using very high concentrations of cells (relative to the amounts we normally employed) to accelerate the formation of tissues. A slew of authors claimed that jacking up the cell count of the stuff you were starting with would net faster tissue formation. Diagram and diagram pointing towards heightened number of cells raising the expression of CAM’s (cell-adhesion molecules). The theory seemed sound enough; the more sticky cells you had the better they stuck together – but it was fairly suspicious that no one was posting hard number. Which CAMs were being activated? By how much? And most importantly why?
I should have trusted my gut there. The squirming, writhing snake of doubt coiling in my insides as I tried something I’d not attempted before. Would have saved me two and half months of glaring down the eyepiece of a microscope, that’s for sure. Every well or petri gummed up within days of plating. You would see the most beautiful islands of cells form; little raft-like colonies serenely collecting in the growth media. Then it all went sour. The rafts wouldn’t connect on the surface. I wanted an archipelago, but all I got were cancerous little atolls. The keratinocytes would form hideous spheres that choked the cells in the center – starved them of the nutrients on the outside. The resulting dead zones would poison everything outward until you had a thin rim of struggling cells, glued to the corpses of their parent and sister generations. It was frustrating – we switched out growth media. We changed cell lines. Didn’t matter – stick too many cells into a plate at once and you were going to get an epidermal traffic jam.
November brought Hayes’ annoyance clip-clopping around the lab. Finding out what doesn’t work is well and fine for academia but the Hindu horse was not after papers. We had several lengthy discussions before I finally decided to change methodology. To appease his interests I switched to using equine cell lines and a growth medium that was more solid-phase2,3. The method was the one I had originally planned to use but due to the amount of time and effort needed had decided against it. But with Hayes breathing down my neck and three months already exhausted, a few weeks on a more reliable method seemed like a better trade off.
I spent the bulk of late November all the way the brink of January perfecting the method. Over the Christmas holiday I stumbled across an exciting publication4 that greatly accelerated our work as well. I ended up shifting away from wet-methods and commandeered our old crystallography preparation glove box to exploit the sterile environment. Ueda’s method called for repeated spraying of pre-cultured cells onto burn victims our skin – however their process was slow and required months of rehabilitation. Why settle for squirts and dabs, hoping things would take root? Why strap your patients down and tell them not to itch their growing skin when you could present them with their new flesh all at once and skip the acclimation? They were trying to play the odds. They knew half the time it would fail and went with a less work intensive method. More chances to succeed would lessen the brunt of the many failures. They strove for statistical significance. But that’s the mistake with thinking academically. You think 7/10 is a good chance of success – it’s only three duds! – but when you’re dealing with rare materials and peoples lives – that’s three chances for things to go wrong. That three chances you’re going to waste your time and attention. I intended to take chance out of the equation.
Solid phase has the advantage that you can do things in rapid sequence and “high” yield. It makes complex chemistry trivial. It lets you rival enzymes in speed and specificity. It’s a multibillion dollar industry that has made it possible to call someone up and order proteins and custom DNA sequences. It also only works on a select number of substrates and can be finicky at that. If the building blocks are available then people will gladly hop right on that for you. If they aren’t.. well then be prepared to start tooling with a system that took the greater part of the four decades to master. You want a methyl group here? Add an extra zero to pricetag. Thankfully, that wasn’t the case in this situation. All they had to do was stick a couple of peptides off the surface off polyacrylate. That was doable enough – you just dipped your desired surface in this solution for an hour, rinsed and repeated as needed. It left us with a microlayer of peptide sticking up off the surface. A painted sheath of molecular Velcro™ that we could stick whatever we wanted onto.
Our initial tests proceeded marvelously. We covered a polyacrylate plate in CAM analogs so the initial seeding of equine keratinocytes would have someplace to stick. No need to worry about rafts or spheres in solution – they’d form a nice even mat where we wanted. After that we just just applied daily mistings of daughter cells to keep thing moving along. Epidermal cells are easy to work with once you get them going; they don’t require a vascular network to suckle nutrients from. No need for complicated artificial blood vessels. Mix your favorite nutrient broth into the daily spritz and they grow happily. It was kind of like gardening really. A nice, thick mat of the pinkest, healthiest flesh you could imagine, sprawling out, eager for more surface space. The procedure and solution prep was simple enough I could even teach it to the other lab staff. By mid-February we were averaging a quarter of a foot of viable, transplantable skin a week. I was due time, and I decided to send Hayes an update.
He ended up coming in earlier today and I could not have been more excited to show off our progress. We had already switched over to a morphic cell line – taken from a patient in a burn unit here in Colorado. She was a wolf of some uncertain descent – although you wouldn’t know it looking at her. Her right hand, arm, shoulder and face had been severely injured from a grease fire. An industrial sized kitchen deep-fryer had caught aflame and her efforts to put it out with an extinguisher had instead splattered sizzling oil all over the place. Namely her. The file photo was grizzly – a wrist and lower arm devoid of fur and bundled tight with oozing gauze. A muzzle deformed by heat, snout sanded down to the bone by a rough licking tongue of heat. One eye was effectively glued shut by her presently fused eyelids. The damage draped down her neck and neck like a sickening shawl of melted flesh. The burn unit had had to perform an emergency skin graft to repair her throat after it became infected and ended up not using all of the flesh they borrowed from her right thigh. Hayes had convinced them to donate it to our project.
The look of revulsion that crumbled his face and bent the tilaka on his brow spoke volumes regarding the success of our work. It is the look of surprise. The mask you wear when gazing into something unexpected. I have seen that look before. I have worn it before. It is what we scientists adorn ourselves when confronted with an unanticipated discovery. A strange crystal in the bottom of our beakers. A curious color change in solution. A napkin sized hunk of skin dangling off a mannequin. Hayes arms folded as he gawked at the glistening, naked flesh I had put on display for him – as if to put a barrier between himself and the product.
“What is this?” he had managed after a moment, to which I was oh so happy to reply,
“It’s her.”
There was an awkward span of disquiet as Hayes struggled to digest the information. To intake the majesty of what our project had accomplished. I offered further elucidation,
“As you can see, Dr. Hayes, we have been able to successfully grow a tissue culture from our candid collaborator. We’ve transferred cultured cells into solution and have been spray coating them onto a surface so that – “
“Well – yes I can see that,” he rudely interrupted, “But, what is it.. mounted on?”
“Oh! Do you like the mannequin? We needed a solid support for the growing tissue and I decided that, rather than use something mundane that a more dynamic surface would suit. As you can see –”
I paused to wriggle my arms into the black neoprene arm ports of the glovebox. The upset in homeostasis caused a hiss and sputter of escaping pressure to billow from a side port. The whoosing wail compelled an air compressor beneath to whirl into motion. The thu-thu-thunking of the piston forced me to raise my voice over the mechanical symphony as I grabbed the sides of the mannequins head and pivoted it to more directly face us.
“ – the keratinocytes take very readily to the surface! Through careful application, we can grow the developing tissue into any structure we want! We transplanted some of the facial tissue and are trying to grow fingers –”
– I motioned to the hand fragment sporting what amounted to a fingerless glove of wolf-skin.
“I recalled from the patient’s photos, she had experienced damage around the eyes? So what I have done here is drawn some lines. We’re going to inject ceramide1 – and that’ll make the cells here engage in apoptosis,” I picked up a pipette to demonstrate, circling around the black-marker spots a few times for emphasis. “..Bam! Instant eye holes.”
Hayes was silent for a while. I considered repeating myself in case he had not heard me over the pump but his look of disgust had not softened. His gaze stretched across the face of the mannequin – before twisting away, as though he could not bear to meet its eyeless stare. Instead, he scrutinized the hand whose frozen grasp awaited the touch of developing skin. I tried to read his expression, but looking over my shoulder, elbows deep in arm ports was neither comfortable nor useful. I could see something whirling in the gears behind those bent mawari ears. He scanned along the fleshless fingers of the hand, musing on it before looking at the pipets and then back up to me.
“How fast is it growing? Do you think you could have an arm’s worth by April?”
“That might be pushing it, but yeah I think so. Do you want me to send you progress shots?”
“No thank you Dr. Fluttertail – I’ll send you measurements for the patient. I’ll meet with you then,
I frowned a bit, watching him leave in a hurry. I rather expected him to be more… excited about the advances we’d made. The specificity of his request was promising, but his tone was off-putting. Wasn’t this exactly what he wanted? He knew how much work had gone into this project. It was hard to imagine something as visually distasteful as skin growing on mannequin could turn the stomach of a morph whose own daughter had half her flesh sizzled off. Perhaps it was a cultural thing – but even so, one really oughtn’t look a gift horse in the mouth.
1] Deshpande M. “Three-dimensional organization of dermal fibroblasts by macromass culture.” Biotechnology and Applied Biochemistry 2008 49(1) 65-72
2] Lima-Neto JF,; Fernandes CB,;, Alvarenga MA,; Golim MA,; Landim-Alvarenga FC. “Viability and cell cycle analysis of equine fibroblasts cultured in vitro.” Cell and Tissue Banking, 2010, 26(5), 365-368
3] Chen C,; Li GF,; Liu W,; Yang NZ,; Wang B,; Zhang C,; Wang ZJ.; “Construction of Tissue-Engineered skin by mix-seeding” Chinese Journal of Plastic Surgery 2010, 26(5), 365-368
4] Ueda M, “Sprayed Cultured mucosal epithelial cell for deep dermal burns,” Journal of Craniofacial Surgery 2010, 21(6), 1792-1832
5] Tarek A.T,; Thomas D.M,; Lina M.O; “A house divided: ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death,” Biochimica et Biophysica Acta 2006 12, 2027-2036
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